Sperm Cytoplasm Research Articles

Characterization of heat shock protein 70 in the red claw crayfish ( Cherax quadricarinatus): Evidence for its role in regulating spermatogenesis

Heat shock protein 70 (HSP70) is a member of molecular chaperones and is important for reproductive biological processes in eukaryotes. In this regard, a full length HSP70 comprised of 2366 nucleotides from the red claw crayfish Cherax quadricarinatus (CqHSP70) was characterized, which with an open-reading frame of 1959 bp encoded 652 amino acid residues. Its mRNA transcript expression in the testes was measured by real-time quantitative PCR. CqHSP70 mRNA transcripts were detected in all organs especially high in the testes. Furthermore, the greatest transcript levels were found during the spermatogonial preparation phase, while the lowest levels were found during the resting stage of the reproductive cycle. In addition, its distribution in the testes was determined by Western blot and immunohistochemistry. Western blot results revealed a single immunoreactive band with an estimated molecular mass of 70 kDa in different phases of testes development. The strongest immunolabeling positive signal was found in spermatogonia, with lower positive staining in secondary spermatocytes, and weak or absent level in the mature sperm. Interestingly, CqHSP70 was mainly located in the cytoplasm of sperm cells. Thus, our results indicate that CqHSP70 is an essential protein in spermatogenesis, with a crucial reproductive function during sperm maturation in invertebrates.

MicroRNA activity in theArabidopsismale germline

Most of the core proteins involved in the microRNA (miRNA) pathway in plants have been identified, and almost simultaneously hundreds of miRNA sequences processed in the Arabidopsis sporophyte have been discovered by exploiting next-generation sequencing technologies. However, there is very limited understanding about potentially distinct mechanisms of post-transcriptional regulation between different cell lineages. In this review the focus is on the Arabidopsis male gametophyte (pollen), where the germline differentiates after meiosis giving rise to the male gametes. Based on comparative analysis of miRNAs identified in sperm cells by in-depth sequencing, their possible functions during germ cell specification and beyond fertilization are discussed. In addition, 25 potentially novel miRNAs processed in sperm cells and pollen were identified, as well as enriched variations in the sequence length of known miRNAs, which might indicate subfunctionalization by association with a putative germline-specific Argonaute complex. ARGONAUTE 5 (AGO5), by close homology to AGO1 and localizing preferentially to the sperm cell cytoplasm in mature pollen, may be part of such a complex.

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca2+ increase occurs as a Ca2+ wave at each sperm entry site in the polyspermic egg. Some Ca2+ waves are preceded by a transient spike-like Ca2+ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca2+ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca2+ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca2+ store for the Ca2+ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.

Acid Extrusion from Human Spermatozoa Is Mediated by Flagellar Voltage-Gated Proton Channel

Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.

Shedding Light on Sperm pHertility

The acquisition of fertilization capacity by sperm is regulated by intracellular pH (pH(i)), but the transport pathways that regulate pH(i) are not well understood. Lishko et al. (2010) now report that Hv1, the voltage-sensitive proton channel, is present in human sperm and is an important regulator of the functional maturation of sperm.

Acid Extrusion from Human Spermatozoa is Mediated by Flagellar Hv1 Proton Channel

Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. After ejaculation, and during their transit through the female reproductive tract, sperm motility is initiated and then hyperactivated to allow the spermatozoa to penetrate through the viscous oviductal mucus and the egg's protective vestments. These processes are known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown due to the inability to measure membrane conductances in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at an unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, and removal of extracellular zinc, a potent Hv1 blocker.Surprisingly, endocannabinoid anandamide was also found to modulate Hv1 activity in the sperm cells as well as in the heterologously expression system. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. In contrast to the large Hv1 current in human sperm cells, the amplitude of the outward current recorded under similar conditions from mouse spermatozoa was 30 times smaller; therefore, mouse sperms seem to have a different mechanism for acid extrusion.In human sperm, Hv1-induced intracellular alkalinisation should activate pH-dependent Ca2+ channel CatSper and control intracellular Ca2+ concentration. Finally, since Hv1 lies upstream in the signalling cascades leading to sperm activation, hyperactivation, and capacitation, Hv1 is an attractive target for the control of male fertility.

Gene expression in the dimorphic sperm cells of Plumbago zeylanica: transcript profiling, diversity, and relationship to cell type

Plumbago zeylanica produces cytoplasmically dimorphic sperm cells that target the egg and central cell during fertilization. In mature pollen, the larger sperm cell contains numerous mitochondria, is associated with the vegetative nucleus (S(vn)), and fuses preferentially with the central cell, forming endosperm. The other, plastid-enriched sperm cell (S(ua)) fuses with the egg cell, forming the zygote and embryo. Sperm expressed genes were investigated using ESTs produced from each sperm type; differential expression was validated through suppression subtractive hybridization, custom microarrays, real-time RT-PCR and in situ hybridization. The expression profiles of dimorphic sperm cells reflect a diverse and broad complement of genes, including high proportions of conserved and unknown genes, as well as distinct patterns of expression. A number of genes were highly up-regulated in the male germ line, including some genes that were differentially expressed in either the S(ua) or the S(vn). Differentially up-regulated genes in the egg-targeted S(ua) showed increased expression in transcription and translation categories, whereas the central cell-targeted S(vn) displayed expanded expression in the hormone biosynthesis category. Interestingly, the up-regulated genes expressed in the sperm cells appeared to reflect the expected post-fusion profiles of the future embryo and endosperm. As sperm cytoplasm is known to be transmitted during fertilization in this plant, sperm-contributed mRNAs are probably transported during fertilization, which could influence early embryo and endosperm development.

The counting and sizing of spermatozoa from ten animal species using a Coulter counter.

Accurately calibrated Coulter Counters, Models ZB Industrial and F, were used to count and size spermatozoa before and after Zaponin treatment which lyses accompanying debris, droplets and peripheral sperm cytoplasm. Sperm specimens from the cauda epididymis of the rabbit, Guinea pig, hamster, rat and mouse were without accompanying particles and could be sized without Zaponin treatment. The large acrosome cap of the guinea pig swelled rapidly when the spermatozoa were released into an isotonic solution and measurement was only possible after equilibrium had been reached. Zaponin treatment completely dissolved rat and hamster spermatozoa within a few seconds and about 50% of the mouse spermatozoa. Spermatozoa form the cauda epididymis of the bull were accompanied by some unspecific debris which made size determination without Zaponin treatment difficult. A separate population of cytoplasmic droplets was not present and the amount of accompanying cytoplasm, as shown by its removal with Zaponin, was the least for the species examined. The size of spermatozoa in ejaculated specimens from the dog varied considerably according to whether the cytoplasmis droplet was still present, but after Zaponin treatment all specimens were about the same size. Ejaculated specimens from the European wild boar contained a separate population of small droplets which were sufficiently different in size from the spermatozoa to allow separate counting and sizing without Zaponin treatment. Ejaculated specimens from the Rhesus monkey required incubation to release the spermatozoa from the clot before they could be counted and sized. Their size tended to vary slightly according to the length of incubation. Ejaculated specimens from the rabbit and from man were so heavily contaminated with debris that counting and sizing was not possible without Zaponin treatment. The relationship between the amount of debris and the numbers of spermatozoa was extremely variable. The debris in human specimens was separated from the spermatozoa by downward fractionation of the motile spermatozoa into increasing concentrations of bovine serum albumin, so allowing measurement of untreated spermatozoa for the first time. The sperm size distribution curves for all the ten species examined, both before and after Zaponin treatment, were positively skewed. The peaks were broader and flatter when Zaponin was not used. Sperm sizes, in terms of total volume and of the diameter of a sphere of that volume, are given for all the species at both the mode and the mean of the size distribution curves. After Zaponin treatment the mean size was between a volume of 15 and 50 mum3 or an equivalent spherical diameter of 3-5 mum. Before Zaponin treatment all the sperm types were greater than 20% larger by volume and the mean volume was between 25 and 190 mum3.

Intracellular calcium oscillations in mammalian eggs at fertilization

Intracellular calcium oscillations in mammalian eggs at fertilization

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P < 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen

Mitochondrial DNA in the male reproductive cells of rapeseed (Brassica napus L.) was monitored by fluorescence microscopy of Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide. Mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen. This result corresponds well with the maternal inheritance of mitochondria in rapeseed determined by previous genetic analyses. To better characterize the mode of inheritance of the mitochondrial linear plasmid in rapeseed, which is transmitted through pollen, we analyzed by indirect immunofluorescence microscopy the expression and localization of ORF6 protein, a putative RNA polymerase encoded by the plasmid. ORF6 protein was expressed in mature pollen and specifically localized in the cytoplasm of sperm cells in the mature pollen. This suggests that the genes encoded by the plasmid DNA are transcribed in the mature pollen by its own RNA polymerase (ORF6 protein) and that the gene expression in the generative cells may be needed for transmission of plasmid DNA through the pollen.

UItrastructure of spermatozoa in the free-living marine nematode Paracanthonchus macrodon (Nematoda, Chromadorida)

Summary The spermatozoa from testes and uteri of the free-living marine nematode Paracanthonchus macrodon (Cyatholaimidae, Chromadorida) were studied with an electron microscope. The spermatozoa from the testes are unpolarized cells covered by numerous filopodia. The sperm nuclei have oval- or bean-shaped outlines and are surrounded by mitochondria. The peripheral cytoplasm is devoid of organelles and contains evenly distributed filamentous material. The filopodium content resembles that of the sperm cytoplasm. A submembrane layer of 13–16 nm thick, longitudinally oriented, microtubule-like fibres (MLF) strengthens the filopodia. The spermatozoa from the uterus are amoeboid cells with the discrete nucleus surrounded by mitochondria. The bulk of the sperm cytoplasm lacks organelles and is filled with MLF, which also underlie the sperm plasmalemma. The spermatozoa develop large pseudopodia which form hemidesmosome-like junctions with the uterus wall. A layer of a subsurface osmiophilic material concentrated at the junction point is associated with MLF, which run out into the cytoplasm as parallel arrays. The unique cytoplasmic components of the nematode spermatozoa, the membranous organelles and fibrous bodies, were not found in the spermatozoa of P. macrodon; this pattern of the spermatozoon structure is interpreted as a highly reduced condition.

Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization.

Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.

Male genital system and spermiogenesis of Nanorchestes amphibius (Acari: Endeostigmata: Nanorchestidae): anatomy, histology, and evolutionary implications.

In the present article the anatomy and histology of the male genital system of an endeostigmatid mite are described for the first time. The Endeostigmata probably are a paraphyletic group supposed to include the most primitive actinotrichid mites. In Nanorchestes amphibius, the testis comprises a paired germinal region connected with an unpaired glandular region. In the germinal region, spermiogenesis takes place in cysts of a somatic cell containing germ cells representing the same developmental stage. In the lumen of the glandular region, the spermatozoa are stored together with secretions of the glandular epithelium. These secretions are probably involved in the formation of spermatophores. From the glandular region, spermatozoa and secretions are released into the vasa deferentia that histologically can be divided into three sections, beginning with a short paired region with strong circular muscles serving as a sphincter, continuing with a paired proximal zone, followed by a short unpaired distal section. The distal vas deferens leads into the chitinous, unpaired ductus ejaculatorius which is followed by the progenital chamber. The ductus ejaculatorius is composed of a proximal section and a proximal, central, and anterior chamber. It is accompanied by a complex system of muscles and sclerites probably involved in the formation and ejaculation of the spermatophore. A similar organization can also be found in Prostigmata, but not in Oribatida. Anterior to the progenital chamber is located a paired accessory gland that probably produces a lipid secretion. Spermiogenesis is characterized by disintegration of the nuclear envelope, condensation of chromatin, and extensive reduction of the amount of sperm cell cytoplasm. The mature aflagellate, U-shaped spermatozoa are simple in structure and lack mitochondria and an acrosome complex. The results do not support the current view that Nanorchestidae are more closely related to Sarcoptiformes, i.e., Oribatida and Astigmata, than to Prostigmata.

Human sperm DNA integrity: correlation with sperm cytoplasmic droplets

Objectives To examine the retention of sperm cytoplasmic droplets (CD) and DNA denaturation (DD) in semen from fertile and infertile men. Methods Semen samples were obtained from consecutive nonazoospermic men presenting for infertility evaluation (n = 101) and fertile men presenting for vasectomy (n = 13). The standard semen parameters (sperm concentration, motility, and morphology), sperm DD, and sperm CD were monitored. Sperm DD was evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA. Results The mean (±SE) percentages of spermatozoa with CD and DD were significantly higher in infertile than in fertile men (sperm CD 15.7% ± 0.8% versus 4.8% ± 0.7% and sperm DD 22.0% ± 1.5% versus 10.8% ± 1.8%, respectively). Sperm CD and DD were positively correlated ( r = 0.59). Also, sperm CD and DD values correlated inversely with the standard semen parameters. Conclusions Our data demonstrate that the retention of sperm CD correlates positively with sperm DD and that significantly higher sperm DD and CD are found in infertile than in fertile men. These data suggest that the enhanced susceptibility of sperm DNA to denaturation is associated with the abnormal disposal of residual sperm cytoplasm in the testis and/or epididymis.

How a Sperm Turns On An Egg

When a sperm joins with an egg, development is begun by a signal encoded in oscillations in the concentration of intracellular free calcium ([Ca2+]i) in the cytoplasm of the egg. Precisely how the interaction of egg and sperm triggers these oscillations in [Ca2+]i has been a matter of debate, but soluble extracts of sperm cells can reproduce the effect. The Ca2+ is released from intracellular stores in response to increased concentrations of inositol 1,4,5-trisphosphate (IP3), suggesting that a phosphatidylinositol-specific phospholipase C, which generates IP3 from phosphoinositides could be the culprit. But known PLCs can't substitute for sperm in activation of the egg. Saunders et al. now report that mouse sperm produce a previously uncharacterized isoform of PLC that they call PLCζ. Eggs microinjected with complimentary RNA encoding PLCζ showed oscillations in [Ca2+]i with temporal characteristics similar to those in normally fertilized eggs. Furthermore, the amount of PLCζ from just one sperm cell (about 50 fg) mimicked activation by a sperm, and depletion of PLCζ from sperm extracts by immunoprecipitation removed egg-activating activity. Thus, PLCζ from the sperm cytoplasm appears to be the key molecular determinant of egg activation. The authors note that their findings may be applied to treatment of male infertility or to egg activation for production of stem cells.C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, F. A. Lai, PLCζ: A sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129, 3533-3544 [Abstract] [Full Text]

How a Sperm Turns On An Egg

When a sperm joins with an egg, development is begun by a signal encoded in oscillations in the concentration of intracellular free calcium ([Ca 2+ ] i ) in the cytoplasm of the egg. Precisely how the interaction of egg and sperm triggers these oscillations in [Ca 2+ ] i has been a matter of debate, but soluble extracts of sperm cells can reproduce the effect. The Ca 2+ is released from intracellular stores in response to increased concentrations of inositol 1,4,5-trisphosphate (IP 3 ), suggesting that a phosphatidylinositol-specific phospholipase C, which generates IP 3 from phosphoinositides could be the culprit. But known PLCs can't substitute for sperm in activation of the egg. Saunders et al. now report that mouse sperm produce a previously uncharacterized isoform of PLC that they call PLCζ. Eggs microinjected with complimentary RNA encoding PLCζ showed oscillations in [Ca 2+ ] i with temporal characteristics similar to those in normally fertilized eggs. Furthermore, the amount of PLCζ from just one sperm cell (about 50 fg) mimicked activation by a sperm, and depletion of PLCζ from sperm extracts by immunoprecipitation removed egg-activating activity. Thus, PLCζ from the sperm cytoplasm appears to be the key molecular determinant of egg activation. The authors note that their findings may be applied to treatment of male infertility or to egg activation for production of stem cells. C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, F. A. Lai, PLCζ: A sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development. Development 129 , 3533-3544 [Abstract] [Full Text]

Parental Background Predisposes Baltic Salmon Fry to M74 Syndrome

Parental Background Predisposes Baltic Salmon Fry to M74 Syndrome

Injection of a Sperm Extract Triggers Egg Activation in the Newt Cynops pyrrhogaster

Unfertilized eggs of the newt Cynops pyrrhogaster are arrested at the second meiotic metaphase. The primary signal for egg activation is a transient increase in [Ca2+]i, which is triggered by the fertilizing sperm and propagates over the egg cortex as a Ca2+ wave. We injected an extract of Cynops sperm (SE) into unfertilized eggs and induced a wave-like [Ca2+]i increase which resulted in activation and resumption of meiosis. The SE-injected eggs showed degradation of cyclin B1 and DNA replication. When SE was boiled or treated with proteinase K before injection, it was unable to cause egg activation. Preinjection of Ca2+-chelator BAPTA before SE injection inhibited egg activation. These results indicate that a heat-labile and proteinaceous factor in the sperm cytoplasm induces a transient increase in [Ca2+]i which is required for egg activation. Injection of IP3 into unfertilized eggs caused an increase in [Ca2+]i and egg activation, but injection of cADP-ribose did not. These results support the hypothesis that Ca2+ release at fertilization occurs via IP3 receptors.

Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa

Objective: To determine whether varicocele is associated with retention of sperm cytoplasmic droplets in infertile men. Design: Retrospective study. Setting: University infertility clinic. Patient(s): Nonazoospermic men with idiopathic (n = 69) and varicocele-associated infertility (n = 73), and 20 fertile controls presenting for vasectomy. Intervention(s): None. Main Outcome Measures(s): Standard semen parameters and percentage of spermatozoa with cytoplasmic droplets on Papanicolaou smears. Result(s): No statistically significant differences were found between the fertile and infertile groups with respect to semen volume. Fertile controls had significantly greater mean percent sperm motility and normal morphology than infertile men. The mean percentage of sperm with residual cytoplasm was statistically significantly different in all three groups. Infertile men with varicocele had the highest percentage of sperm with cytoplasmic droplets, the next highest level being in men with idiopathic infertility and the lowest level in fertile controls (11.7 ± 1.0, 8.1 ± 0.9 and 3.2 ± 0.4%, respectively, P<.0001). Conclusion(s): Our data show that idiopathic and even moreso, varicocele-related male infertility are conditions associated with impaired disposal of residual sperm cytoplasm by the testis and/or epididymis. These data provide a possible mechanism for the observed semen abnormalities and reduced fertility potential associated with varicocele and idiopathic male infertility.