Permeabilized Sperm Research Articles

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5'-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.

Effects of the czech propolis on sperm mitochondrial function.

Propolis is a natural product that honeybees collect from various plants. It is known for its beneficial pharmacological effects. The aim of our study was to evaluate the impact of propolis on human sperm motility, mitochondrial respiratory activity, and membrane potential. Semen samples from 10 normozoospermic donors were processed according to the World Health Organization criteria. Propolis effects on the sperm motility and mitochondrial activity parameters were tested in the fresh ejaculate and purified spermatozoa. Propolis preserved progressive motility of spermatozoa in the native semen samples. Oxygen consumption determined in purified permeabilized spermatozoa by high-resolution respirometry in the presence of adenosine diphosphate and substrates of complex I and complex II (state OXPHOSI+II) was significantly increased in the propolis-treated samples. Propolis also increased uncoupled respiration in the presence of rotenone (state ETSII) and complex IV activity, but it did not influence state LEAK induced by oligomycin. Mitochondrial membrane potential was not affected by propolis. This study demonstrates that propolis maintains sperm motility in the native ejaculates and increases activities of mitochondrial respiratory complexes II and IV without affecting mitochondrial membrane potential. The data suggest that propolis improves the total mitochondrial respiratory efficiency in the human spermatozoa in vitro thereby having potential to improve sperm motility.

MARCKS Protein Is Phosphorylated and Regulates Calcium Mobilization during Human Acrosomal Exocytosis

Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.

2020 INSL3 RECEPTOR RXFP2 IS EXPRESSED ON THE SPERM ACROSOME

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20112020 INSL3 RECEPTOR RXFP2 IS EXPRESSED ON THE SPERM ACROSOME Daniel DaJusta, Candace F. Granberg, Yi Wang, Shaohua Zhang, Kent Hamra, and Linda A. Baker Daniel DaJustaDaniel DaJusta Dallas, TX More articles by this author , Candace F. GranbergCandace F. Granberg Dallas, TX More articles by this author , Yi WangYi Wang Dallas, TX More articles by this author , Shaohua ZhangShaohua Zhang Dallas, TX More articles by this author , Kent HamraKent Hamra Dallas, TX More articles by this author , and Linda A. BakerLinda A. Baker Dallas, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2248AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Insulin-3 (INSL3) and its receptor, RXFP2 (aka LGR8), are key mediators in testicular descent due to their action on the fetal gubernaculum. Insl3 or Rxfp2 knockout mice demonstrate bilateral intra-abdominal cryptorchidism and infertility. The RXFP2 receptor mRNA has been demonstrated in pachytene spermatocytes, but the function and localization of the protein in spermatogenesis has not been further documented. We sought to investigate whether adult sperm express the RXFP2 receptor. METHODS Sperm were extracted from the cauda epididymis of reproductive age WT mice and from human ejaculated sperm. Sperm in standard medium was capacitated with calcium and sodium bicarbonate for 1 hour. To assess permeabilized (fixed) sperm, capacitated sperm were air dried on slides for 20 minutes, fixed in 4% paraformaledhyde and washed in PBS. Anti-RXFP2 rabbit polyclonal antibody 1:150 was incubated followed by red fluorescent anti-rabbit donkey antibody 1:1000. Green fluorescent acrosome staining was performed with FITC labeled Anti-Peanut Agglutinin antibody (PNA-FITC) followed by DAPI blue nuclear counterstaining. To assess non-permeabilized (live) sperm, capacitated sperm were centrifuged and resuspended in PBS with anti-RXFP2 rabbit polyclonal antibody 1:150. Again, sperm were centrifuged and resuspended in PBS with anti-rabbit donkey antibody 1:1000. PNA-FITC was then added for acrosome staining. Finally, 50μl of the sperm solution was slide mounted with DAPI counterstain. Control experiments omitted the primary antibody. Images were acquired with a Nikon Eclipse fluorescent microscope using NIS-Elements Br 2.30 software. RESULTS The acrosomes of capacitated adult WT mice sperm and human ejaculated sperm in both permeabilized and non-permeabilized state stained positive for RXFP2 (Figure 1). CONCLUSIONS The RXFP2 protein is localized to the acrosome of mature spermatozoa in mice and human. Given this observation, we speculate a possible new role for the INSL3-RXFP2 signaling system in acrosomal functions. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e807-e808 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniel DaJusta Dallas, TX More articles by this author Candace F. Granberg Dallas, TX More articles by this author Yi Wang Dallas, TX More articles by this author Shaohua Zhang Dallas, TX More articles by this author Kent Hamra Dallas, TX More articles by this author Linda A. Baker Dallas, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Regulation of tyrosine kinase activity during capacitation in goat sperm

Protein tyrosine phosphorylation is a key event accompanying sperm capacitation. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. It is necessary to differentiate the localization of the tyrosine-phosphorylated proteins in spermatozoa to understand the link between the different phosphorylated proteins and the corresponding regulated sperm function. cAMP plays a pivotal role in the regulation of tyrosine phosphorylation. The intracellular cAMP levels were raised in goat spermatozoa by the addition of the phosphodiesterase inhibitor, IBMX in conjugation with caffeine. Tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents. Treatment of caudal spermatozoa with IBMX and caffeine, time dependent up-regulated phosphorylation of the protein of molecular weights 50 and 200 kDa was observed. Increased phosphorylation was observed with a combination of IBMX and caffeine treatment. Tyrosine phosphorylation in caput spermatozoa was not affected significantly under these conditions. The expression level of tyrosine kinase in sperm was examined with specific inhibitors and with anti-phosphotyrosine antibody. The indirect immunofluorescence staining was carried out on ethanol permeabilized sperm using anti-phosphotyrosine antibody. Western blot analysis was done using two separate PKA antibodies: anti-PKA catalytic and anti-PKA RIalpha. Almost no difference was found in the intracellular presence of the PKA RIalpha and RIIalpha subunits in caput and caudal epididymal spermatozoa. However, the catalytic subunit seemed to be present in higher amount in caudal spermatozoa. The results show that caprine sperm displays an enhancement of phosphorylation in the tyrosine residues of specific proteins under in vitro capacitation conditions.

PTP1B Dephosphorylates N-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

Acrosomal exocytosis, a special type of regulated secretion

The acrosome is a single secretory granule present in the head of mammalian--and other animal groups--sperm. Secretion of this granule is an absolute requirement for physiological fertilization. Acrosome exocytosis is a synchronized and tightly regulated all-or-nothing process, with no recycling of membranes. In the last few years, it has been shown that acrosomal exocytosis is mediated by a molecular mechanism that is homologous to that reported in the secretion of neuroendocrinal cells. Moreover, because of its particular characteristics, acrosomal exocytosis is a unique mammalian model for the study of the different steps of the membrane fusion cascade. Combining results in intact and permeabilized sperm, the following sequence of events has been proposed. In resting sperm, SNARE proteins are locked in inactive cis complexes. Sperm activation causes a calcium increase in the cytoplasm that promotes the production of cAMP and activates Rab3A. Afterwards, NSF and alphaSNAP disassemble cis complexes and the free SNAREs are then able to reassemble in loose trans complexes. Membrane fusion is arrested at this stage until calcium is released from inside the acrosome by inositol 1,4,5-trisphosphate-sensitive calcium channels to trigger the final steps of membrane fusion, which require fully assembled trans SNARE complexes and the calcium sensor synaptotagmin. This working model is still incomplete and tentative. Its improvement will be important to share light on this and other processes of regulated exocytosis. Moreover, it will bring new perspectives into the field of sperm-related fertility and sterility.

Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.

Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCβII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain – a key region for the function of synaptotagmins – are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.

Haprin, a Novel Haploid Germ Cell-specific RING Finger Protein Involved in the Acrosome Reaction

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.

The role of F‐actin cytoskeleton‐associated gelsolin in the guinea pig capacitation and acrosome reaction

The acrosomal reaction (AR) is a regulated sperm exocytotic process that involves fusion of the plasma membrane (PM) with the outer acrosomal membrane (OAM). Our group has described F-actin cytoskeletons associated to these membranes. It has been proposed that in regulated exocytosis, a cortical cytoskeleton acts as a barrier that obstructs membrane fusion, and must be disassembled for exocytosis to occur. Actin-severing proteins from the gelsolin family have been considered to break this barrier. The present study attempted to determine if gelsolin has a function in guinea pig sperm capacitation and AR. By indirect immunofluorescence (IIF), gelsolin was detected in the apical and postacrosomal regions of the head and in the flagellum in both capacitated and non-capacitated guinea pig spermatozoa. By Western blotting, gelsolin was detected in isolated PM and OAM of non-capacitated spermatozoa. Gelsolin and actin were detected in a mixture of PM-OAM obtained by sonication, and both proteins were absent in membranes of capacitated spermatozoa. Inhibition of three different pathways of PIP2 hydrolysis during capacitation did not cancel gelsolin loss from membranes. Gelsolin was detected by Western blotting associated to membrane cytoskeletons obtained after phalloidin F-actin stabilization and Triton-X treatment; additionally, by immunoprecipitation, it was shown that gelsolin is associated with actin. By electron microscopy we observed that skeletons disassemble during capacitation, but phalloidin prevents disassembly. A three-dimensional skeleton was observed that apparently joins PM with OAM. Exogenous gelsolin stimulates AR assayed in a permeabilized spermatozoa model. Results suggest that gelsolin disassembles F-actin cytoskeletons during capacitation, promoting AR.

Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.

When boar spermatozoa are incubated in a medium designed for in vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim of the present study is to examine the role of cytoplasmic free Ca(2+) in boar sperm agglutination induced by a cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, and resuspended in a modified Krebs-Ringer-Hepes solution lacking calcium chloride. The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 minutes and were then used to determine the percentages of head-to-head agglutinated spermatozoa. Percentages of head-to-head agglutinated spermatozoa in the samples rose significantly after incubation, from 28% to 61%-62%, after adding to the medium a cell-permeable, phosphodiesterase-resistant cAMP analogue (cBiMPS, 10 microM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus a cell-permeable phosphodiesterase inhibitor (IBMX, 25 microM). However, the promoting effects of these reagents were blocked when spermatozoa were pretreated with a cell-permeable Ca(2+) chelator (BAPTA-AM, 25 microM), whereas the same pretreatment with a cell-impermeable Ca(2+) chelator (BAPTA, 25 microM) had almost no influence on sperm agglutination. Adding thapsigargin, a potential Ca(2+)-ATPase inhibitor, to the medium raised the percentages of agglutinated spermatozoa in a concentration-dependent manner for concentrations up to 4 microM. When 4 microM thapsigargin and 10 microM cBiMPS were examined for their effects on free Ca(2+) levels in sperm heads by using a cell-permeable Ca(2+) indicator (fluo-3/AM), the samples incubated with both or either of these reagents contained many head-to-head agglutinated cells that exhibited intense fluorescence in the heads. In control samples incubated without these reagents by contrast, most spermatozoa were free (unagglutinated) cells and characterized by almost no or only slight fluorescence in the heads. Moreover, morphological observation of Giemsa-stained preparations revealed that most agglutinated spermatozoa possessed darkly stained acrosomes, which distinguished them from acrosomereacted spermatozoa. This indicated that the sperm agglutination was not a result of the acrosome reaction. Furthermore, with indirect immunofluorescence of Ca(2+)-ATPases, the mouse monoclonal antibody to this enzyme demonstrated high affinity to the acrosomes of permeabilized spermatozoa. Based on these results, we conclude that cytoplasmic free Ca(2+) is involved in sperm head-to-head agglutination induced by a cAMP analogue.

Transgenic approaches to retinal development and function in Xenopus laevis

The African clawed frog Xenopus laevis has long been used to study the development and function of the vertebrate retina. An efficient technique for generating transgenic Xenopus embryos, the REMI procedure, has enabled the stable overexpression of transgenes in developing and mature X. laevis. In the retina, transgenes driven by retinal-specific promoters have been used to study protein trafficking, circadian rhythms, and retinal degeneration. The REMI technique is surprisingly simple, consisting of integration of plasmid DNA into permeabilized sperm nuclei, followed by transplantation of these nuclei into unfertilized eggs. Here, we describe the reagents and steps necessary for generation of transgenic embryos using the REMI reaction and discuss its applications for the study of retinal development.

A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6, the mammalian orthologue of Chlamydomonas PF16.

cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.

Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction

The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.

Post-testicular development of a novel membrane substructure within the equatorial segment of ram, bull, boar, and goat spermatozoa as viewed by atomic force microscopy

Atomic force microscopy has been used to investigate changes in the plasma membrane overlying the head region of mammalian spermatozoa (bull, boar, ram, goat, stallion, mouse, and monkey) during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle. On ejaculated ram, bull, boar, and goat spermatozoa the postacrosomal plasma membrane has a more irregular surface than that covering the acrosome. The equatorial segment, by contrast, is relatively smooth except for an unusual semicircular substructure within it that has a coarse uneven appearance. This substructure (referred to as the equatorial subsegment) is situated adjacent to the boundary between the postacrosomal region and the equatorial segment itself and seems to be confined to the order Artiodactyla as it has not been observed on stallion, mouse, or monkey spermatozoa. The equatorial subsegment develops during epididymal maturation, and following induction of the acrosome reaction with Ca 2+ ionophore A23187, its topography changes from a finely ridged appearance to that resembling truncated papillae. A monoclonal antibody to the equatorial subsegment binds only to permeabilized spermatozoa, suggesting that the subsegment is related to the underlying perinuclear theca that surrounds the sperm nucleus. A role for the equatorial subsegment in mediating fusion with the oolemma at fertilization is discussed.

Immunization of female cynomolgus macaques with a synthetic epitope of sperm-specific lactate dehydrogenase results in high antibody titers but does not reduce fertility.

Previous studies have reported reduced fertility in female baboons immunized with a synthetic peptide derived from the sperm-specific isozyme of lactate dehydrogenase (LDH-C). In this study, a similar approach was used to immunize female cynomolgus macaques with the same peptide sequence (bC5-19) conjugated to a T-cell epitope from tetanus toxin (TT). Twelve female monkeys were immunized with bC5-19:TT delivered with Ribi MPL adjuvant vehicle, and 10 control female monkeys were injected with the adjuvant vehicle only. All 12 females in the treatment group developed LDH-C-specific serum antibodies as measured by ELISA, but anti-LDH-C antibodies were not detected in vaginal fluids of the immunized animals. After 4 months of timed mating immediately following the immunizations, five of the ten immunized females became pregnant, as did six of the ten control females. Anti-sera from both pregnant and nonpregnant bC5-19:TT-immunized females recognize a single band at 35 kDa on Western blots of whole sperm extracts, and purified Igs from the same sera localize along the principle piece of the flagellum of permeabilized sperm.

Synaptotagmin VI Participates in the Acrosome Reaction of Human Spermatozoa

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.