Sperm Midpiece Research Articles

223 DESCRIPTION OF GENITALIA AND SPERM RECOVERED POSTMORTEM FROM A PYGMY SPERM WHALE, KOGIA BREVICEPS

A pygmy sperm whale (Kogia breviceps; adult male; 350 kg) was stranded and died on a beach near Cabo Bustos (Asturias, North of Spain) on March 12th, 2005. Finding specimens of this species is a rare event on Spanish shores, although this whale is not considered endangered. Postmortem examination was performed 24 h later. Genitalia (testicles and epididymides) were extracted. The postmortem report indicated that vas deferens and seminal glands seemed to contain an important amount of semen, which was not recovered. Refrigerated genitalia were send to our laboratory, arriving around 40 h postmortem. The refrigerated testicles were in poor physical condition upon arrival, indicating advanced tissue detoriation. The epididymides (very long) were not closely attached to the testicles, but were connected by a loose conjunctive membrane. We divided the epididymides into four regions that approximated the (1) caput, (2) mid-region, (3) corpus, and (4) cauda. Physical characteristics of the genitalia are described in Table 1. The left testicle was larger, and possibly more active, than the right one. A sperm sample was obtained from the cauda region after incising the tissue. Osmolality and pH of the sample were 428 mOsm/kg and 6.62, respectively (maybe due to tissue breakdown) and the sperm concentration was 1194 × 106/mL. Spermatozoa were immotile, even after diluting in buffered medium; it is possible that postmortem damage occurred quickly. However, using flow cytometry we determined that 57% of cauda spermatozoa had intact plasma membranes and acrosomes (determined by staining with 37 mmol/mL propidium iodide and 1 μg/mL PNA-FITC; Sigma, Madrid, Spain). Examination by phase contrast microscopy (×600) showed many spermatozoa with abnormal heads and bent midpieces and flagella, even in the cauda (13% and 21%, respectively). Sperm head morphometry was studied using DiffQuick staining and an automated analysis system (SCA2000; Microptic, Barcelona, Spain). Mean sperm head size was 3.71 ± 0.19 × 2.61 ± 0.12 μm in width and length, respectively. Computer analysis (AnalySiS-GmbH, Cologne, Germany) of phase contrast images revealed that the mean size of the sperm midpiece and flagellum were 3.44 ± 0.19 and 40.95 ± 2.02 μm, respectively. The information obtained after postmortem recovery of the testes and epididymis should be useful to future conservation efforts of the pygmy sperm whale and similar species. The rapid deterioration of the testicular tissue by 40 h postmortem was not expected since good quality sperm samples have been obtained at similar postmortem intervals in other species. Therefore, we recommend that postmortem sperm recovery should be accomplished as rapidly as possible in this species. Table 1. Genitalia measurements

Acetylcholine causes an increase of intracellular calcium in human sperm

Sperm nicotinic acetylcholine receptors (nAChRs) can influence motility and the initiation of acrosome reaction (AR). We report that AR initiation by acetylcholine (ACh) in capacitated human sperm requires both Na+ and Ca2+ in the external medium. Pre-incubation with 50 microM 3-quinuclidinyl benzilate (QNB) or 50 nM strychnine failed to inhibit the ACh-initiated AR, demonstrating that muscarinic AChRs and nAChRs containing alpha9 subunits do not mediate this event. Choline (2.5, 5 and 10 mM), a highly specific but low potency agonist of the alpha7 nAChR initiated AR, with its effect blocked by the nAChR antagonist methyllycaconitine (MLA). ACh (50-400 microM) stimulated a small transient rise in the intracellular Ca2+ in sperm populations loaded with FURA-2, with 200 microM ACh being maximal (146 nM +/- 23 SEM). The nAChR antagonists, alpha-bungarotoxin (alpha-BTX) and MLA, reduced the ACh-initiated Ca2+ rise by 75 and 78%, respectively, demonstrating the majority of the rise is mediated through nAChRs containing alpha7 or alpha9 subunits. Single cell imaging studies using FLUO-3 resolved two patterns of ACh-stimulated Ca2+ increase in the sperm head: 94% of responding sperm displayed a rise (59.6% +/- 5.7 SEM increase from resting fluorescence intensity), returning to resting levels over a period of 2-3 min. The remaining sperm (6%) displayed a sharp spike of Ca2+ ( approximately 1 min; 86% +/- 4.3 SEM change in fluorescence intensity), followed by abrupt loss of fluorescence, a pattern suggestive of AR. A Ca2+ influx in the sperm midpiece appeared to accompany the Ca2+ influx seen in the head. These observations confirm an ionotropic role for nAChRs in sperm function.

Sperm competition and the evolution of sperm midpiece volume in mammals

AbstractSperm competition occurs when the gametes of two, or more, males compete for opportunities to fertilize a given set of ova. This study examined whether mammalian sperm morphology could be affected by sexual selection. Comparisons were made of linear and volumetric measurements of the sperm head, midpiece and flagellum for 123 species representing 71 genera of mammals having either single partner mating systems or multiple partner mating systems. Sperm midpiece volume was found to be significantly larger in those species were females mate with more than one male during a single peri‐ovulatory period. A positive correlation was also found between relative testes sizes and sperm midpiece volumes, while no relationship was found with any other sperm measure. These results indicate that, within mammals, the evolution of sperm midpiece volume has been affected by selection pressures resulting from sperm competition. The significance of these findings may relate to the density of mitochondria within the sperm midpiece. The mitochondria provide the energy required for sperm motility, in the absence of glycolytic support. A higher mitochondrial loading may therefore be associated with enhanced sperm motility.

Nicotinic Acetylcholine Receptor Subunits and Associated Proteins inHuman Sperm

We demonstrated previously the involvement of a nicotinic acetylcholine receptor containing an alpha7 subunit in the human sperm acrosome reaction (a modified exocytotic event essential to fertilization). Here we report the presence in human sperm of alpha7, alpha9, alpha3, alpha5, and beta4 nicotinic acetylcholine receptor subunits and the following proteins known to be associated with the receptor in the somatic cell: rapsyn and the tyrosine kinases c-SRC and FYN. The alpha7 subunit appears to exist as a homomer in the posterior post-acrosomal and neck regions of sperm and is probably linked to the cytoskeleton via rapsyn. The alpha3, alpha5, and beta4 subunits are present in the sperm flagellar mid-piece of sperm and possibly exist as alpha3alpha5beta4 and/or alpha3beta4 channels. The alpha9 subunit is present in the sperm mid-piece. We detected the FYN and c-SRC tyrosine kinases in the flagellar mid-piece region. Both co-precipitated only with the nicotinic acetylcholine receptor beta4 subunit. Immunolocalization with a C-terminal SRC kinase antibody, which recognizes several members of SRC kinase family, detected a SRC kinase co-localized with the alpha7 subunit in the neck region of sperm. Immunoprecipitation studies with that antibody demonstrated that the alpha7 subunit is associated with a SRC kinase. Antagonists of tyrosine phosphorylation inhibited the acetylcholine-initiated acrosome reaction, suggesting the involvement of a SRC kinase in the acrosome reaction.

Mice Deficient in CHRNA7, a Subunit of the Nicotinic Acetylcholine Receptor, Produce Sperm with Impaired Motility1

In this study we investigate the role of the CHRNA7 subunit (also known as the alpha7 subunit) of the nicotinic acetylcholine receptor in mouse sperm function. We confirm by reverse-transcription-polymerase chain reaction the expression in adult mouse testis of Chrna7 mRNA and demonstrate the subunit's presence in mouse sperm by immunoblot. Alpha-bungarotoxin binds a range of nicotinic acetylcholine receptor subunits, including the CHRNA7 subunit. Localization studies using a fluorescent alpha-bungarotoxin-tetramethyl-rhodamine conjugate revealed specific binding sites on the midpiece of mouse sperm with fainter alpha-bungarotoxin binding on the remainder of the flagellum. Mice engineered with a double-null disruption of the Chrna7 gene displayed only faint fluorescence on the midpiece, suggesting that the CHRNA7 contributed the majority of the observed alpha-bungarotoxin binding sites. The location of alpha-bungarotoxin binding suggested that nicotinic acetylcholine receptors may play an ionotropic role in sperm motility. Sperm from Chrna7(-/-) mice display no difference in number, morphology, viability or spontaneous acrosome reaction rate compared with Chrna7(+/+) sperm. Studies using computer-assisted sperm analysis indicate the motility of Chrna7(-/-) sperm is significantly impaired. This impairment is characterized by significantly reduced swimming velocities, failure to maintain vigorous swimming, and lower levels of hyperactivated swimming patterns in Chrna7(-/-) sperm compared with Chrna7(+/+) sperm. This is the first genetic evidence that sperm nicotinic acetylcholine receptors are important for maintenance of normal sperm motility.

MtDNA Investigations After Differential Lysis

In order to gain information regarding the ethnic origin of an unknown offender in a murder case it was necessary to sequence the HV1 and 2 regions of the mitochondrial DNA (mtDNA). The only evidentiary material that could be linked to the suspect was DNA, extracted from spermatozoa after differential lysis. The observed mtDNA sequences were identical to the sequences of the victim. Therefore, we had to check if this was a coincidence or the result of a technical limitation of the testing procedure. Subsequently, we performed a systematic study. In cases with complete separation of sperm and female cells it wasn't possible to obtain a mtDNA sequence for the sperm fraction. This phenomena is based on the loss of the sperm's flagellum and mid-piece during the first lysis step and a concomitant loss of the sperm's mitochondria. In our murder case, a minor carry-over of female cells to the sperm fraction was responsible for the sequencing result.

Microanatomy Of Hedylopsis Ballantinei, A New Interstitial Acochlidian Gastropod From The Red Sea, And Its Significance For Phylogeny

This is the first description of an acochlidian gastropod species from the Red Sea. More than 20 specimens of Hedylopsis ballantinei n. sp. have been extracted from subtidal coral sand in the Gulf of Aqaba. As a model organism for this poorly known, highly aberrant and enigmatic opisthobranch group, the anatomy of this new species is described in detail. Major organ systems have been reconstructed from histological semithin serial sections. The central nervous system has also been studied by means of immunocytochemical staining and confocal laser scanning techniques; dorsal bodies and rhinophoral ganglia are described for the first time for Acochlidia. Hardparts were examined using SEM, the midpiece of an acochlidian sperm is described with aid of TEM for the first time. Our results show Hedylopsis ballantinei n. sp. to be similar to the European H. spiculifera (junior synonym H. suecica). Differences include tropical occurrence, larger rhinophoral ganglia, different arrangement of ampulla and genital glands, and the probable absence of anterior male genitalia. Hedylopsis ballantinei n. sp. plays an important role in understanding acochlidian phylogeny, since it combines an array of morphological features that appear plesiomorphic for Acochlidia, e.g. a (small) mantle cavity, a unidirectional stomach lacking any cuticular elements, a tube-like, holohepatic digestive gland, a prepharyngeal, epiathroid central nervous system with separate cerebral, pleural and pedal ganglia, dorsal bodies associated with the cerebral ganglia, a (short) visceral loop with three ganglia, a monaulic and hermaphroditic reproductive system, and elongate spiral sperm. On the other hand, the absence of anterior male genitalia appears to be a derived condition that is shared with microhedylacean species. The aberrant possession of three glycogen helices in the sperm midpiece appears unique for opisthobranchs. With present, limited knowledge, the Acochlidia is regarded as a monophyletic group that is supported by several autapomorphies, with a hypothetical origin from acteonoidean opisthobranchs, i.e. diaphanid-like ancestors.

Selenium and endocrine systems

The trace element selenium (Se) is capable of exerting multiple actions on endocrine systems by modifying the expression of at least 30 selenoproteins, many of which have clearly defined functions. Well-characterized selenoenzymes are the families of glutathione peroxidases (GPXs), thioredoxin reductases (TRs) and iodothyronine deiodinases (Ds). These selenoenzymes are capable of modifying cell function by acting as antioxidants and modifying redox status and thyroid hormone metabolism. Se is also involved in cell growth, apoptosis and modifying the action of cell signalling systems and transcription factors. During thyroid hormone synthesis GPX1, GPX3 and TR1 are up-regulated, providing the thyrocytes with considerable protection from peroxidative damage. Thyroidal D1 in rats and both D1 and D2 in humans are also up-regulated to increase the production of bioactive 3,5,3'-tri-iodothyronine (T3). In the basal state, GPX3 is secreted into the follicular lumen where it may down-regulate thyroid hormone synthesis by decreasing hydrogen peroxide concentrations. The deiodinases are present in most tissues and provide a mechanism whereby individual tissues may control their exposure to T3. Se is also able to modify the immune response in patients with autoimmune thyroiditis. Low sperm production and poor sperm quality are consistent features of Se-deficient animals. The pivotal link between Se, sperm quality and male fertility is GPX4 since the enzyme is essential to allow the production of the correct architecture of the midpiece of spermatozoa. Se also has insulin-mimetic properties, an effect that is probably brought about by stimulating the tyrosine kinases involved in the insulin signalling cascade. Furthermore, in the diabetic rat, Se not only restores glycaemic control but it also prevents or alleviates the adverse effects that diabetes has on cardiac, renal and platelet function.

Germ Cell Nuclear Factor Relieves cAMP-response Element Modulator τ-mediated Activation of the Testis-specific Promoter of Human Mitochondrial Glycerol-3-phosphate Dehydrogenase

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle that transfers reduction equivalents from the cytosol into the mitochondrion. Within the testis, immunohistological analysis localized human mGPDH to late spermatids and to the midpiece of spermatozoa. The expression of human mGPDH is regulated by two somatic promoters, and here, we describe a third testis-specific promoter of human mGPDH. The usage of this testis-specific promoter correlates with the expression of a shortened mGPDH transcript of approximately 2.4 kb in length, which is solely detectable from testicular RNA. Within the testis-specific promoter, we detected a cAMP-response element (CRE) site at -51, which binds the testis-specific transcriptional activator CRE modulator tau (CREMtau) in electrophoretic mobility shift assays. This recognition site overlaps with a nuclear receptor binding half-site at -49, which binds the testis-specific transcriptional repressor germ cell nuclear factor (GCNF). Both factors compete for binding to the same DNA response element. Ectopic expression of CREMtau in HepG2 cells activated a promoter-driven luciferase construct in transient transfection experiments. Additional cotransfection of GCNF relieved this activity, suggesting a down-regulation of CREMtau-mediated activation by GCNF. This effect was preserved by introducing the CRE/nuclear receptor-binding element into a heterologous promoter context. Our data suggest a down-regulation of CREMtau-mediated gene expression by GCNF, which might be a general regulation mechanism for several postmeiotically expressed genes with a temporal expression peak during early spermatid development.

Effects of Sexual Selection Upon Sperm Morphology and Sexual Skin Morphology in Primates

We review possible effects of sexual selection upon sperm morphology, and sexual skin morphology, in primates. Comparative morphometric studies, involving 31 species representing 21 primate genera, revealed a positive relationship between volume of the sperm midpiece, occurrences of multiple partner matings by females, and large relative testes sizes, which indicate sperm competition. The midpiece houses the mitochondria required to power sperm motility. Hence, sperm competition may have influenced the evolution of increased mitochondrial loading in species where females mate with multiple partners during the fertile period. Females of some Old World monkey species and female chimpanzees exhibit large estrogen-dependent sexual skin swellings during the follicular phase of the menstrual cycle. Studies of mandrills support the conclusion that swellings act primarily as sexually attractive, graded signals and that swelling size may indicate current reproductive quality. Measurements of the genitalia in chimpanzees indicate a secondary function for female swellings. The swelling increases the operating depth of the female's vagina by ≤50% during the fertile phase of her cycle. Males have evolved long, filiform penes capable of placing sperm close to the os cervix during competitive multipartner matings. This may exemplify how morphologic specializations in females can influence the coevolution of advantageous genitalic specializations in males: the phenomenon that Eberhard (1985) dubbed cryptic female choice.

Vitamin D: Is there a role in male infertility?

The role of vitamin D in male infertility has been implicated by several animal studies. Deficiencies of vitamin D have been shown to retard spermatogenesis in animal models. The biological activity of vitamin D is mediated by the vitamin D receptor (VDR). VDR interacts with promoter regions of target genes in several cell types including Sertoli cells. VDR null mutant mice have significant gonadal insufficiencies. The presence of VDR in human reproductive tissue and the role of vitamin D in male infertility have not yet been demonstrated. We investigated the presence of VDR in human sperm. We performed a prospective evaluation of sperm from fertile and infertile men for qualitative and semi-quantitative analysis of VDR expression. Semen specimens were collected from 11 fertile and 20 infertile men based on their semen parameters as defined by the WHO/Kruger criteria and by the length of time attempting conception. Qualitative analysis for VDR was performed by immunohistochemistry using a monoclonal antibody to human VDR. Immunoprecipitation of equal amounts of total sperm protein lysates followed by immunoblotting with VDR antibodies was performed to semi-quantitate the VDR levels. VDR expression was measured against sperm motility and concentration. Immunohistochemical analysis of semen samples demonstrated the presence of VDR on the mid-piece of sperm. Immunoprecipitation followed by immunoblotting with VDR antibody resulted in 2 possible forms of VDR (50 KDa and 40KDa). Infertile men with sperm concentrations <5 million/cc (n=10) showed approximately 3-fold increase in the 50kDa VDR expression versus the fertile controls. Infertile men with concentrations >5 million/cc (n=10) had approximately 2-fold increase in the 50kDa VDR expression versus the fertile controls. A similar trend was noted for the 40kDa VDR protein. We also observed a downward trend in VDR expression for patients with low motility, irrespective of fertility status. Our results demonstrate the presence of VDR on human sperm for the first time. In addition, we showed VDR expression was inversely related to sperm concentration in infertile men versus fertile controls. Finally we observed a linear trend for motility and VDR expression but these data reflect preliminary results and our studies are ongoing. The mechanism of action of vitamin D through the VDR on sperm has yet to be determined. Our data suggest a role for vitamin D in sperm physiology and male infertility.

Rat kinesin light chain 3 associates with spermatid mitochondria

We recently discovered that in rat spermatids, kinesin light chain KLC3 can associate with outer dense fibers, major sperm tail components, and accumulates in the sperm midpiece. Here, we show that mitochondria isolated from rat-elongating spermatids have bound KLC3. Immunoelectron microscopy indicates that the association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the plasma membrane to the developing midpiece. KLC3 is able to bind in vitro to mitochondria from spermatids as well as somatic cells employing a conserved kinesin light chain motif, the tetratrico-peptide repeats. Expression of KLC3 in fibroblasts results in formation of large KLC3 clusters close to the nucleus, which also contain mitochondria: no other organelles were present in these clusters. Mitochondria are not present in KLC3 clusters after deletion of KLC3's tetratrico-peptide repeats. Our results indicate that the rat spermatid kinesin light chain KLC3 can associate with mitochondria.

Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa.

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.

Hamster contraception associated protein 1 (CAP1).

Based on cDNA and amino acid sequence, we demonstrate that hamster contraception associated protein 1 (CAP1) protein (an homolog of DJ-1 in mouse, CAP1/SP22/RS in rat and DJ-1/RS in human) is conserved during evolution. Through solubilization studies, it was demonstrated that hamster CAP1 has a peripheral membrane localization. SDS-PAGE analysis revealed that the migration pattern for hamster CAP1 compared to the other rodent counterparts, rat and mouse was different; indicating species-specific differences in the protein (possibly due to post-translational modifications). This protein also shows a ubiquitous presence in both somatic and germ tissues, and has been localized to the sperm tail. It was noticed that hamster CAP1 was lost from the mid piece of spermatozoa during capacitation. Interestingly, following in vitro treatment with ornidazole, CAP1 was lost from the spermatozoa and immunofluorescence studies showed that the major loss was from the mid piece of the spermatozoa. Another interesting feature highlighted about hamster CAP1 is its tendency to exist in two pI isoforms. Summarily, hamster CAP1 appears to exhibit species-specific differences compared to its rodent counterparts with respect to its unique peripheral localization, its size, two pI isoforms, and fate during capacitation, which may have implications in its functions.

A dominant-negative isoform of hypoxia-inducible factor-1 alpha specifically expressed in human testis.

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1. Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe. Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria. In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta. However, hHIF-1alphaTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity.

Pregnancy from intracytoplasmic sperm injection of a sperm head and detached tail

Objective To evaluate sperm ultrastructure and the efficacy of intracytoplasmic sperm injection (ICSI) with careful positioning of the sperm midpiece next to the sperm head in a patient with easily decapitated sperm syndrome. Design In vitro fertilization case report with retrospective sperm ultrastructure analysis. Setting In vitro fertilization clinic and andrology laboratory. Patient(s) A couple seeking treatment for diminished ovarian reserve and male factor infertility using donor oocyte IVF. Intervention(s) Motile sperm inadvertently decapitated during micromanipulation were used for a modified ICSI procedure in which the midpiece was carefully positioned proximal to the sperm head. Sperm were also analyzed by transmission electron microscopy (TEM). Main outcome measure(s) Fertilization rate, embryo development, pregnancy, and the incidence of normal sperm ultrastructure. Result(s) The ICSI resulted in a fertilization rate of 63% and embryo development of good to moderate quality of 36% of the embryos. Transfer of two embryos resulted in a pregnancy and birth of a healthy child. Normal sperm centrosomes were identified retrospectively. Conclusion(s) Pregnancy can result in patients where the sperm are decapitated during immobilization before ICSI if normal centrosomes are present and the head and midpiece are positioned closely together.

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.

Male reproductive toxicity of trichloroethylene: sperm protein oxidation and decreased fertilizing ability.

The objective of the present study was to characterize and investigate potential mechanisms for the male reproductive toxicity of trichloroethylene (TCE). Male rats exposed to TCE in drinking water exhibited a dose-dependent decrease in the ability to fertilize oocytes from untreated females. This reduction in fertilizing ability occurred in the absence of treatment-related changes in combined testes/epididymides weight, sperm concentration, or sperm motility. In addition, flow cytometric analysis showed that there were no treatment-related differences in sperm mitochondrial membrane potential or acrosomal stability. TCE caused slight histological changes in efferent ductule epithelium, coinciding with the previously reported ductule localization of cytochrome P450 2E1. However, no alterations were noted in the testis or in any segment of the epididymis. Because there were no treatment-related changes to sperm indices and no clear pathological lesions to explain the reduced fertilization, the present study investigated TCE-mediated sperm oxidative damage. Oxidized proteins were detected by immunochemical techniques following the derivatization of sperm protein carbonyls with dinitrophenyl hydrazine. Immunochemical staining of whole, intact sperm showed the presence of halos of oxidized proteins around the head and midpiece of sperm from TCE-treated animals. The presence of oxidized sperm proteins was confirmed by Western blotting using in vitro-oxidized sperm as a positive control. Thiobarbituric acid reactive substances analyses showed a dose-dependent increase in the level of lipid peroxidation in sperm from treated animals, as well. Oxidative damage to sperm may explain the diminished fertilizing capacity of exposed animals and provide another mechanism by which TCE can adversely affect reproductive capabilities in the male.

Identification of Glutamate Receptors and Transporters in Mouse and Human Sperm

gamma-Aminobutyric acid (GABA) and glutamate (Glu) are considered as the predominant inhibitory and excitatory neurotransmitters in mammalian central nervous systems (CNS), respectively. The presence of the GABA system and metabotropic glutamate receptors in sperm prompted us to explore the existence of ionotropic glutamate receptors and glutamate transporters in sperm. Immunofluorescent analysis was used to investigate the existence and location of glutamate, glutamate receptor (NR2B), and glutamate transporter (GLT1) in mouse and human sperm. Our present results showed that NR2B was located in the midpiece of sperm, whereas GLT1 mainly existed in the head. Moreover, glutamate uptake activity was detected in mouse sperm and it could be blocked by dihydrokainic acid (DHK, GLT1-selective inhibitor) and DL-threo-beta-hydroxyaspartic acid (THA, nonselective inhibitor). In addition, reverse transcription-polymerase chain reaction technique and sequencing analysis revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6, and KA2) existed in mouse sperm as well as in human sperm. The present findings are the first direct evidence for the existence of ionotropic glutamate receptors and glutamate transporters in sperm. It also indicates that, in sperm, glutamate receptors and transporters might have functions other than neurotransmission.

277.The role of pp60c-src tyrosine kinase in sperm capacitation

A protein kinase A-dependent tyrosine phosphorylation pathway in mammalian spermatozoa has been demonstrated to exist, and is unique to this cell type. As PKA is incapable of directly phosphorylating substrates on tyrosine residues, much research has focused on the identification of an intermediary tyrosine kinase which can be activated by serine/threonine phosphorylation via PKA. Inhibitory studies using genistein, tryphostin, erbstatin and herbimycin A, have demonstrated that the src family of kinases may be responsible for the tyrosine phosphorylation events seen during capacitation (1). Although one src family member, c-yes, has been implicated as the kinase responsible for these events (2), this has since been disputed (3). Another src family member, pp60c-src, can be activated by phosphorylation on Ser-17 by cAMP-dependent protein kinase and Ser-12 by calcium-phospholipid dependent protein kinase C (4), and may be the intermediary tyrosine kinase of interest. Western blot analysis demonstrated the presence of pp60c-src in rat sperm samples isolated from the caput and cauda epididymis. Furthermore, co-immunoprecipitation studies revealed a number of pp60c-src-associated proteins including outer dense fibre 2 (ODF2) and A-kinase anchoring protein 4 (AKAP4). Interestingly, both of these proteins become phosphorylated during capacitation of mouse sperm (data not shown) and AKAP4 is tyrosine phosphorylated in capacitated human sperm (5). These data implicate pp60c-src kinase activity in the phosphorylation of a number of sperm midpiece proteins, which may regulate hyperactivation during capacitation. Further research focusing on the activity of pp60c-src in non-capacitated and capacitated sperm will be conducted. (1) Yaciuk P., et al. (1989) Mol. Cell Biol. 9, 2453–2461. (2) Baker M., et al. (2003) J. Cell Sci. 117, 211–222. (3) Leclerc P., Goupil S (2002) Biol. Reprod. 67, 301–307. (4) Bajpai M., et al. (2003) Arch. Androl. 49, 229–246. (5) Ficarro S., et al. (2003) J. Biol. Chem. 278, 11�579–11�589.