Role Of Sperm Research Articles

Endocannabinoids and Reproduction

Infertility is a worldwide reproductive health problem whose consequences have deep psychological and social impact in health, demographic change, and wellbeing. Thus, the knowledge of basic, conserved modulators of reproduction might contribute to the discovery of new potential target for the exploitation of drugs to treat infertility in humans. Besides the well known effects of endocannabinoids in the control of pain and visceral functions, in the last decade the deep involvement of endocannabinoid system in the control of reproductive functions in both males and females emerged. In fact, endocannabinoids, endogenous lipids that bind to cannabinoid receptors, modulate reproductive axis at both central and local level. Endocannabinoid signalling is critical for gonadotropin release and sex steroid biosynthesis, for the formation of functional male and female gametes, for fertilization, preimplantation embryo development, implantation, and postimplantation embryonic growth, and for labouring delivery as well. Endocannabinoids are also involved in the neuroendocrine control of reproduction functions through the modulation of stress, food intake, appetite, and sexual behaviour. Recently, new roles in sperm “startup” and gamete quality emerged and impairment of the physiological endocannabinoid tone and signalling has been reported in clinical cases of human infertility. Our hope is that this special issue may be important and timely since a deep knowledge of endocannabinoid system in reproduction might open new perspectives in clinical applications, pointing to endocannabinoid signalling as a novel target for correcting infertility, and for improving reproductive health in humans. The papers submitted to this special issue in the International Journal of Endocrinology take into account the multifaceted aspects of reproduction. Basic and evolutionarily conserved mechanisms of endocannabinoid activity in reproduction have also been included. E. Cottone et al. submitted an extensive review on the role of the endocannabinoids in the central regulation of reproduction in nonmammalian vertebrates, especially fish and amphibian; they correlate the morphofunctional distribution of cannabinoid receptors to key molecules involved in the control of reproductive functions, such as Gonadotropin Releasing Hormone (GnRH), dopamine, aromatase, and pituitary gonadotropins. The role of the endocannabinoid system as an ancient signalling system, that has been evolved over 500 million years, is highlighted in the paper of R. Chianese et al. that reports the presence of endocannabinoids in a nonmammalian model, the anuran amphibian Rana esculenta, and the functional crosstalk between these bioactive lipids and the GnRH system, shedding light on their different regulation in the brain and in the testis. In particular, a new role for vanilloid receptor emerged in the modulation of testicular GnRH system (both ligands and receptors) providing evidence that an opposite regulation occurs via type-1 cannabinoid receptor and vanilloid receptor signalling. The paper of G. Cacciola et al. is a very interesting and comprehensive review on the pivotal role played by type-1 cannabinoid receptor in spermiogenesis and on its involvement in the chromatin remodelling process that might affect negatively the sperm quality. The emerging evidences on estrogen activity in sperm quality are deeply detailed in a knock-out animal model, opening new intriguing perspectives in the clinical practice for the treatment of male infertility. The deep involvement of endocannabinoid signalling in driving the neurophysiological outcomes of mating behaviours has been reported in the research article submitted by J. M. Stuart et al. By means of lipidomic techniques, this group demonstrates that the levels of endocannabinoids, prostaglandins, and N-acylethanolamines rapidly change in specific brain areas in relationship to different mating strategies providing evidence that the endogenous cannabinoid systems is rapidly modified in response to changes in environment. In the context of the signalling events of pregnancy, B. M. Fonseca et al. presented the most recent progress on the endocannabinoid regulatory functions during decidualization and placentation. They summarized that while the endocannabinoid machinery was found to be expressed in decidual and placental tissues, aberrant endocannabinoid signalling was associated with pregnancy disorders, highlighting the content that the endocannabinoid signalling is a potential player coordinating successful decidualization and placentation. All of these papers have illustrated the potential regulatory interactions of sex steroid hormones with the endogenous cannabinoid system and how they allow reproduction to optimally function. The outstanding review by T. Ayakannu et al. that details the latest understandings of how sex steroids and the endogenous cannabinoid system work synergistically in a variety of cancers truly illustrates how the malfunctions of these signalling mechanisms can have dire effects. Using data from prostate, breast, and endometrial cancers, the review from Konje's group provides compelling evidence that to understand how to fix pathophysiology we must work towards understanding basic functioning physiology in the first place. Rosaria Meccariello Natalia Battista Heather B. Bradshaw Haibin Wang

Fluorescence Technologies for Evaluating Male Gamete (Dys)Function

For many years, andrologists have sought ways of assessing sperm fertility, especially of new sires entering the breeding chain. As knowledge of the complex processes that enable sperm to fertilize eggs has increased, it has become clearer that quantitative estimation of the fertilizing potential of a sire or an ejaculate is actually unlikely ever to be fully realized. Here, we propose that a better approach is to identify substandard males and semen samples. During the past decades, the use of fluorescence technologies in biomedical science has burgeoned, with the development of very powerful instrumentation such as confocal microscopy and flow cytometers of ever-increasing capabilities together with a vast range of fluorochromes and fluorochrome conjugates. This technology has been applied to andrology but thus far in only a relatively simple way. In this review, we offer strategies for assessing a large range of sperm functions thought to be related to fertilizing ability over a temporal window rather than at a single time point. From such an assessment profile, sperm samples that over-respond or do not respond sufficiently could be identified, termed dysfunctional and rejected. We outline the rationales behind such tests, present information on new potentially useful fluorochromes and current flow cytometer models that would be suitable for the multicolour multifunctional tests we propose, and we offer suggestions as to how andrologists might design such multicolour tests for themselves.

Optimisation of total RNA isolation method from bird sperm

The aim of the current study was to determine the optimal RNA extraction in sperm, to support further molecular applications determining the biological roles of sperm in chicken reproduction. Generally, the major issue with RNA isolation is low quantity of RNA is sperm cells. The recent research done on sperm indicates that RNA isolation protocols are specific to the species. RNA extractions from chicken sperm samples (n=47) were respectively performed using TRIzol ® method in combination with PureLink RNA Mini Kit (TRIzol ® Plus RNA Purification System, Invitrogen) and column-based RNA isolation method (PureLink TM RNA Mini Kit, Invitrogen). Both methods were used according to the manufacturer’s protocol but with small modifications. DNase treatment (PureLink ® DNase Set, Invitrogen) was performed in order to eliminate contaminating genomic DNA from samples. To test the quantity and quality the isolated total RNA was checked by the NanoDrop (Thermo Scientific, Wilmington, DE). In general, higher yields were obtained by pure column based isolation when compared to combined TRIzol ® and column based approach. The mean of the yields of total RNA samples ranged from 36.93 ± 2.8 ng (TRIzol ® ) to 381.77 ± 191.3 ng (PureLink TM ). All RNA samples had spectrophotometry values between 1.40 – 1.45 (TRIzol ® ); 1.78 – 1.90 (PureLink TM ) for absorbance ratios A260/A280, and between 0.52 – 1.21 (TRIzol ® ); 1.38 – 2.23 (PureLink TM ) for ratios A260/A230. These results demonstrate that the most suitable protocol of total RNA extraction from bird sperm is column-based RNA isolation method and should be further tested in studies on fertility in birds.

RABL-regulated pathways: a new tale in sperm function

RABL-regulated pathways: a new tale in sperm function

Comprehensive mapping of the bull sperm surface proteome

While the mechanisms that underpin maturation, capacitation, and sperm-egg interactions remain elusive it is known that these essential fertilisation events are driven by the protein complement of the sperm surface. Understanding these processes is critical to the regulation of animal reproduction, but few studies have attempted to define the full repertoire of sperm surface proteins in animals of agricultural importance. Recent developments in proteomics technologies, subcellular fractionation, and optimised solubilisation strategies have enhanced the potential for the comprehensive characterisation of the sperm surface proteome. Here we report the identification of 419 proteins from a mature bull sperm plasma membrane fraction. Protein domain enrichment analyses indicate that 67% of all the proteins identified may be membrane associated. A large number of the proteins identified are conserved between mammalian species and are reported to play key roles in sperm-egg communication, capacitation and fertility. The major functional pathways identified were related to protein catabolism (26S proteasome complex), chaperonin-containing TCP-1 (CCT) complex and fundamental metabolic processes such as glycolysis and energy production. We have also identified 118 predicted transmembrane proteins, some of which are implicated in cell adhesion, acrosomal exocytosis, vesicle transport and immunity and fertilisation events, while others have not been reported in mammalian LC-MS-derived sperm proteomes to date. Comparative proteomics and functional network analyses of these proteins expand our system's level of understanding of the bull sperm proteome and provide important clues toward finding the essential conserved function of these proteins.

Proteomic discovery of diverse immunity molecules in mammalian spermatozoa

Ongoing proteomic analyses are providing a wealth of new data on the composition of the sperm proteome across a range of mammals and other taxa. Although molecular evolution and functional genomic analyses of the proteome have only begun recently, we now broadly understand the molecular composition of sperm. Systems level analysis has revealed a variety of molecular insights into sperm evolution and function, including a remarkable diversity of immunity-related proteins within the proteome. Using existing mammalian sperm proteomes as a starting point, we provide an overview of this important class of sperm proteins and what is known about their function in sperm maturation, sperm quality, sperm competition, and fertilization. The recent observation that many sperm immunity proteins are rapidly evolving, presumably under the influence of positive selection, suggests that they may be responding not only to selection associated with host immunity defense but also with pleiotropic functions in sperm. In addition to the documented role of sperm in the mediation of female immune response, we propose that the fundamental mechanisms involved in cell-cell recognition and binding in both immune processes and fertilization may underlie the multi-functionality of proteins in immunity and reproductive systems.

Assessment of Intracellular Ca2+, cAMP and 1,2‐Diacylglycerol in Cryopreserved Buffalo (Bubalus bubalis) Spermatozoa on Supplementation of Taurine and Trehalose in the Extender

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.

The Use of Anti-VDAC2 Antibody for the Combined Assessment of Human Sperm Acrosome Integrity and Ionophore A23187-Induced Acrosome Reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca2+ transmembrane transport.

Ceramides with 2-hydroxylated, very long-chain polyenoic fatty acids in rodents: From testis to fertilization-competent spermatozoa

Sphingolipids from rodent testis and spermatozoa are known to contain non-hydroxylated (N-) and 2-hydroxylated (2-OH) very-long-chain polyunsaturated fatty acids (VLCPUFA). In this study, the contribution of species with each type of fatty acids to the total ceramides (Cer) and sphingomyelins (SM) was investigated in rat and mouse testis and in rat spermatozoa. The major VLCPUFA in both lipids of testis were N- and 2-OH versions of 28:4n−6, 30:5n−6 and 32:5n−6 in the rat, and predominantly of 30:5n−6 in the mouse. Absent altogether from rat pre-puberal testes, SM and Cer with N-VLCPUFA appeared 10 days earlier than those with 2-OH VLCPUFA in postnatal development, in association with germ cell differentiation. Conversely, in adult fertile rats that were gradually deprived of germ cells in vivo after treatment with doxorubicin, SM and Cer with N-VLCPUFA decreased earlier than their 2-OH counterparts, and neither was present in aspermatogenic testes. In rat epididymal spermatozoa, the content of Cer prevailed over that of SM and 2-OH VLCPUFA prevailed over N-VLCPUFA in both lipids. In mature gametes, the acrosomal reaction resulted in an almost complete hydrolysis of the species of SM that contain both types of VLCPUFA to produce the corresponding Cer. Ceramides are biosynthetic precursors of SM in the testis, but themselves final products in spermatozoa. VLCPUFA-rich SM and Cer are thus produced in germ cells with the teleological objective of fulfilling their ultimate physiological role in spermatozoa that are apt and ready to fertilize an oocyte.

Molecular cloning of pig ZPBP2 and mRNA expression of ZPBP1 and ZPBP2 in reproductive tracts of boars

Zona pellucida binding proteins (ZPBPs) are important receptors of zona pellucida on sperm and play an essential role in sperm–egg interaction. In the present study, the full-length coding regions of transcript variant 1 and 2 of ZPBP2 were cloned in the pig. ZPBP1 gene expression was specifically present in testes of boar reproductive tracts; while ZPBP2 mRNA was detected with highly in testes, moderate in seminal vesicles and low in caudal epididymes. We also examined the expression profile of porcine ZPBP1 and ZPBP2 in testes and seminal vesicles at different developmental stages. The results revealed that ZPBP1 mRNA was not detectable until day 70 and kept relative stable from days 70 to 105, then increased sharply from days 105 to 160 during sexual maturity ( P < 0.01). ZPBP2 mRNA was detected with relative low level until day 70 and but increased significantly from days 70 to 105 ( P < 0.01), and increased further from days 105 to 160 during sexual maturity ( P < 0.01). The data indicated that testes are the main origin of ZPBPs and the expression of ZPBPs are parallel with reproduction development.

ZP-binding peptides identified via phage display stimulate production of sperm antibodies in dogs

Zona pellucida (ZP) glycoproteins play a central role in sperm–oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs.

Spermatogenesis‐defective (spe) mutants of the nematode Caenorhabditis elegans provide clues to solve the puzzle of male germline functions during reproduction

In most species, each sex produces gametes, usually either sperm or oocytes, from its germline during gametogenesis. The sperm and oocyte subsequently fuse together during fertilization to create the next generation. This review focuses on spermatogenesis and the roles of sperm during fertilization in the nematode Caenorhabditis elegans, where suitable mutants are readily obtained. So far, 186 mutants defective in the C. elegans male germline functions have been isolated, and many of these mutations are alleles for one of the approximately 60 spermatogenesis-defective (spe) genes. Many cloned spe genes are expressed specifically in the male germline, where they play roles during spermatogenesis (spermatid production), spermiogenesis (spermatid activation into spermatozoa), and/or fertilization. Moreover, several spe genes are orthologs of mammalian genes, suggesting that the reproductive processes of the C. elegans and the mammalian male germlines might share common pathways at the molecular level.

Sperm–egg adhesion and fusion in mammals

Fertilisation is an orchestrated, stepwise process during which the participating male and female gametes undergo irreversible changes, losing some of their structural components while contributing others to the resultant zygote. Following sperm penetration through the egg coat, the sperm plasma membrane fuses with its oocyte counterpart, the oolemma. At least two plasma membrane proteins essential for sperm-oolemma fusion--IZUMO and CD9 on the male and female gametes, respectively--have been identified recently by classical cell biology approaches and confirmed by gene deletion. Oolemma-associated tetraspanin CD81, closely related to CD9, also appears to have an essential role in fusion. Additional proteins that may have nonessential yet still facilitating roles in sperm-oolemma adhesion and fusion include oolemma-anchored integrins and oocyte-expressed retroviral envelope proteins, sperm disintegrins, and sperm-borne proteins of epididymal origin such as CRISP1 and CRISP2. This review discusses these components of the gamete fusion mechanism within the framework of gamete structure, membrane biology, cell signalling and cytoskeletal dynamics, and revisits the topic of antipolyspermy defence at the oolemma level. Harnessing the mechanisms of sperm-egg fusion is of importance to animal biotechnology and to human assisted fertilisation, wherein male patients with reduced sperm fusibility have been identified.

Identification of Protein Tyrosine Phosphatases and Dual-Specificity Phosphatases in Mammalian Spermatozoa and Their Role in Sperm Motility and Protein Tyrosine Phosphorylation1

Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was rapid and reversible. Pervanadate also increased tyrosine phosphorylation of different proteins in capacitated and noncapacitated spermatozoa. Results showed that the phosphatases PTPN11, DUSP4, and DUSP3 are present in boar, stallion, and dog spermatozoa. PTPRB is also present in boar and stallion spermatozoa but was not detected in dog. The subcellular distribution of the identified phosphatases is diverse, suggesting that they likely have specific roles in sperm. Finally, PTP activity has a positive role in the regulation of motility and is involved in protein tyrosine phosphorylation in mammalian sperm.

Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa

Protein tyrosine phosphorylation is one of the main processes associated with sperm activation. Although this process and its targets have been well characterized, only few tyrosine kinases have been identified so far and their roles in spermatozoa are still largely unknown. In this study, we report the presence and localization of Src kinase in ejaculated human spermatozoa and investigate its role in regulating the processes underlying sperm activation. Specific anti-Src antibodies, against different epitopes of the protein, identified a single band of approximately 70 kDa relating to a protein which is mainly localized in the post-acrosomal region of the head, neck and midpiece. By immunoprecipitation and immunofluorescence techniques performed with antibodies against Src phosphorylated at Tyr416, which identifies the active kinase, we showed an increased phosphorylation during sperm capacitation. Blocking Src activity with SU6656 resulted in a significant reduction in the protein tyrosine phosphorylation. Moreover, this inhibitor also blocked the progesterone-induced acrosome reaction and interfered with the calcium response to progesterone evaluated in fura-2-loaded spermatozoa. No effect on sperm motility and hyperactivation resulted from incubation with SU6656. We identified a novel Src isoform in human spermatozoa, which appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation and acrosome reaction.

Spermatozoa modulate epididymal cell proliferation and protein secretion in vitro

Normal epididymal function, such as protein expression and secretion, is primarily regulated by testicular androgens and temperature. However, the role of spermatozoa in this critical process has never been studied. In order to determine whether sperm itself could regulate epididymal function, we have developed a cell culture system of bovine epididymal cells to study the interactions between spermatozoa and the epididymal epithelium. Primary cells from caput, corpus, and cauda epididymal tissues were cultured in the presence of androgens at 32 degrees C (scrotal) and 37 degrees C (abdominal). Newly synthesized proteins were metabolically labeled with (35)S-methionine after sperm co-incubation and the pattern of secreted proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proliferation rate, protein secretion rate and electrophoretic patterns of secreted proteins were evaluated 48 hr post-co-incubation. Incubation at 32 degrees C indicated that spermatozoa stimulation increases the level of protein secretion of cultured cells from all epididymal sections while it slightly decreases proliferation of corpus cells. At 37 degrees C, spermatozoa co-incubation significantly decreases the protein secretion rate of cultured cells from all epididymal sections. Independently of cell incubation temperature, spermatozoa stimulation induces both an increase in the intensity of radiolabeled proteins and the appearance of new secreted proteins of caput cells without affecting the protein pattern of corpus or cauda cells. Incubation at 37 degrees C, however, greatly modifies the pattern of proteins expressed at 32 degrees C by cauda cells. Taken together, these results support the hypothesis that spermatozoa themselves affect epididymal cell function, most importantly for caput epididymides.

Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

CatSperβ, a Novel Transmembrane Protein in the CatSper Channel Complex

Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca(2+)-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shock protein 70-2, a testis-specific chaperone, and CatSperbeta, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSperbeta was restricted to testis and localized to the principal piece of the sperm tail. CatSperbeta protein is absent in CatSper1(-/-) sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSperbeta is the first identified auxiliary protein to the CatSper channel.

Concerted Evolution Within the Drosophila dumpy Gene

We have determined by reverse Southern analysis and direct sequence comparisons that most of the dumpy gene has evolved in the dipteran and other insect orders by purifying selection acting on amino acid replacements. One region, however, is evolving rapidly due to unequal crossing over and/or gene conversion. This region, called "PIGSFEAST," or PF, encodes in D. melanogaster 30-47 repeats of 102 amino acids rich in serines, threonines, and prolines. We show that the processes of concerted evolution have been operating on all species of Drosophila examined to date, but that an adjacent region has expanded in Anopheles gambiae, Aedes aegypti, and Tribolium castaneum, while the PF repeats are reduced in size and number. In addition, processes of concerted evolution have radically altered the codon usage patterns in D. melanogaster, D. pseudoobscura, and D. virilis compared with the rest of the dumpy gene. We show also that the dumpy gene is expressed on the inner surface of the micropyle of the mature oocyte and propose that, as in the abalone system, concerted evolution may be involved in adaptive changes affecting Dumpy's possible role in sperm-egg recognition.

A role for sperm in regulation of egg-laying in the Nematode C. elegans

BackgroundIn insects and in mammals, male sperm and seminal fluid provide signaling factors that influence various aspects of female physiology and behavior to promote reproductive success and to compete with other males. It is less apparent how important such signaling is in the context of a self-fertile hermaphrodite species. We have addressed this question in the nematode Caenorhabditis elegans, which can reproduce either by hermaphrodite self-fertilization or by male-hermaphrodite mating.ResultsWe have studied the egg-laying defective mutant, egl-32, and found that the cellular basis of the egl-32 egg-laying phenotype is likely a defect in sperm. First, the time of egl-32 action coincides with the timing of spermatogenesis in the hermaphrodite. Second, egl-32 interacts with genes expressed in sperm. Third, mating experiments have revealed that wild-type sperm can rescue the egg-laying defect of egl-32 mutant animals. Most importantly, introduction of mutant egl-32 sperm into wild-type hermaphrodites or females is sufficient to induce an egg-laying defective phenotype.ConclusionPrevious work has revealed that C. elegans sperm release factors that stimulate oocyte maturation and ovulation. Here we describe evidence that sperm also promote egg laying, the release of embryos from the uterus.