Post-Testicular Sperm Maturation: Centriole Pairs, Found in Upper Epididymis, are Destroyed Prior to Sperm's Release at Ejaculation.

The fertilizing sperm's lengthiest unchartered voyage is through the longest, least-investigated organ in a man's body - the Epididymis. Over six meters long in men, ~80 meters in stallions and over one-hundred times a mouse's body length, there are few functions known aside from sperm storage and nutrition. While spermatogenesis is completed in the testes, here we demonstrate sperm centriole reduction occurs within the epididymis. Investigations of GFP-CENTR mice and controls demonstrate both the presence of centriole pairs in the upper caput region of the epididymis and, the destruction, first, of the distal and, then, of the proximal centriole as the sperm transits to the cauda and vas deferens in preparation for its climactic release. These centrioles can neither recruit γ-tubulin nor nucleate microtubules when eggs are inseminated or microinjected, yet numerous maternally-nucleated cytasters are found. These sperm centrioles appear as vestigial basal bodies, destroyed in the mid-to-lower corpus. Post-testicular sperm maturation, in which sperm centrioles found in the caput are destroyed prior to ejaculation, is a newly discovered function for the epididymis.

Presence of N-ethyl maleimide sensitive factor (NSF) on the acrosome of mammalian sperm.

We have extended previous observations to show that the ATPase N-ethyl maleimide sensitive factor (NSF) an important regulator of membrane trafficking and fusion in somatic cells, is present on bovine, murine and rhesus macaque sperm. However, NSFs main effectors, alfa- and beta-SNAP, although present in the developing acrosome, could not be detected in the mature organelle. The fact that NSF localizes mainly to the acrosome suggests that this protein, together with other factors such as rabs and SNAREs, may be a common feature in the triggering/regulation of membrane merging during the mammalian acrosome reaction.

Intracytoplasmic sperm injection: stiletto conception or a stab in the dark.

To describe the importance of molecular and cellular analyses in intracytoplasmic sperm injection (ICSI) the authors review the literature on biological challenges in ICSI and associated techniques. Several matters can be proposed in molecular and cellular challenges in ICSI for safety and efficacy: (1) a reliable and convenient animal model for understanding the molecular and cellular basis of human ICSI must be established, and molecular and cellular analysis of the first cell cycle of human fertilization should be better understood; (2) a proper assay for human sperm function that contributes to the indication for ICSI should be developed; and (3) de novo and transmitted genetic security in ICSI should be examined.

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL.

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.

Arrest of cell cycle progression during first interphase in murine zygotes microinjected with anti-PCM-1 antibodies.

To investigate the function of the centrosome protein PCM-1, antibodies against PCM-1 were microinjected into either germinal vesicle stage meiotic oocytes or fertilized mouse eggs, and cell cycle progression events (i.e., microtubule assembly, chromosome and centrosome organization, meiotic maturation) were assayed. These studies determined that microinjected PCM-1 antibodies arrested cell cycle progression, with anti-PCM-1 arresting fertilized eggs at the pronucleate stage when injected during G1. Analysis of the injected eggs determined that centrosome disruption and microtubule cytaster disorganization accompanied the cell cycle arrest. Anti-PCM-1 blocked neither pronuclear centration, completion of mitosis when microinjected into zygotes at G2, nor meiotic maturation when microinjected into immature oocytes. These results identify a novel role for PCM- 1 in cell cycle regulation, and indicate that PCM-1 must fulfill an essential function for cells to complete interphase.

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin.

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.

Fertilization imaged in 2-, 3- and four dimensions: molecular insights for treating infertility.

Fertilization in humans follows a complex series of events including binding of the sperm to the oocyte plasma membrane, oocyte activation, the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei and the restoration of the sperm centrosome. This duplicates and separates, forming two mitotic spindle poles upon which the parental genomes can intermix to complete fertilization. The use of intracytoplasmic sperm injection (ICSI) has been highly effective as a treatment for severe male infertility and thousands of ICSI babies have been born world-wide. Working with rhesus monkey gametes, we have developed a preclinical animal model for understanding the cell biological basis of ICSI. Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear structures normally removed at the oolemma during in vitro fertilization. These unusual modifications raise concerns that the ICSI procedure itself might lead to the observed increase in chromosome anomalies reported for

Membrane trafficking machinery components associated with the mammalian acrosome during spermiogenesis.

Active trafficking from the Golgi apparatus is involved in acrosome formation, both by delivering acrosomal contents to the nascent secretory vesicle and by controlling organelle growth and shaping. During murine spermiogenesis, Golgi antigens (giantin, beta-COP, golgin 97, mannosidase II) are detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgi-acrosome flow may be relatively unselective, with Golgi residents retrieved before spermiation is complete. Treatment of spermatogenic cells with brefeldin A, a drug that causes the Golgi apparatus to collapse into the endoplasmic reticulum, disrupted the Golgi in both pachytene spermatocytes and round spermatids. However, this treatment did not affect the acrosomal granule, and some beta-COP labeling on the acrosome of elongating spermatids was maintained. Additionally, N-ethylmaleimide sensitive factor, soluble NSF attachment proteins, and homologues of the t-SNARE syntaxin and of the v-SNARE VAMP/synaptobrevin, as well as members of the rab family of small GTPases, are associated with the acrosome (but not the acrosomal granule) in round and elongated spermatids. This suggests that rab proteins and the SNARE machinery for membrane recognition/docking/fusion may be involved in trafficking during mammalian acrosome biogenesis.

A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis.

The normal structure and function of sperm are prerequisites for successful fertilization and embryonic development, but little is known about how defective sperm are eliminated during mammalian spermatogenesis. Here, we describe a ubiquitin-dependent, sperm quality control mechanism that resides in the mammalian epididymis, the site of sperm maturation and storage. We used immunofluorescence, electron microscopy, western blotting and pulse-chase experiments to show that ubiquitin is secreted by the epididymal epithelium and binds to the surface of defective sperm. Most of the ubiquitinated sperm are subsequently phagocytosed by the epididymal epithelial cells. A portion of defective sperm escapes phagocytosis and can be found in the ejaculate. Cultured epididymal cells maintain their ability to produce ubiquitin and phagocytose the defective sperm, as well as the ubiquitin-coated microspheres, in vitro. The surprising phenomenon of cell-surface ubiquitination in defective sperm provides a possible mechanism for sperm quality control in mammals and a new marker of semen abnormalities in men and animals.

Ubiquitin-based sperm assay for the diagnosis of male factor infertility.

Sperm morphology is correlated with fertility in men, yet the existing, subjective sperm morphology assays provide only a limited insight into patients' infertility. Here, we provide the experimental background for a new, objective and automated semen assay, based on the cross-reactivity of defective human spermatozoa with antibodies against a proteolytic marker peptide, ubiquitin. Using immunofluorescence and flow cytometry, we screened the spermatozoa from 17 infertility patients and two fertile donors for their cross-reactivity with anti-ubiquitin antibodies. Thirteen out of 17 patients, but neither of two fertile donors, displayed an increased binding of anti-ubiquitin antibodies to sperm surface, that reflected the occurrence of abnormalities in these samples and was corroborated by available clinical data. Highest correlation coefficient (r = -0.432) was obtained with the cleavage rate after IVF. The contribution of male factor was revealed in several couples previously diagnosed with idiopathic infertility. Ubiquitin-cross-reactive sperm-surface proteins thus seem to be a universal marker of semen abnormalities, including sperm head and tail defects and semen contaminants such as spermatids, leukocytes and cellular debris. We propose that the sperm-ubiquitin tag immunoassay (SUTI) may be a valuable diagnostic tool in treatment of male factor and idiopathic infertility.

Transgenic monkeys produced by retroviral gene transfer into mature oocytes.

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.

Subcellular localization of beta1,4-galactosyltransferase on bull sperm and its function during sperm-egg interactions.

The process of sperm-oocyte recognition is a complex interaction between the plasma membrane of sperm and the extracellular matrix of the oocyte. The best studied mammalian system is the mouse, in which sperm plasma membrane receptors recognize specific oligosaccharides on the egg coat glycoprotein ZP3. A well-defined ZP3 receptor on mouse sperm is beta1,4-galactosyltransferase (GalT). In this study, we investigated the possibility that GalT is present on bull sperm, and that it may participate during bovine sperm-oocyte binding. Using Western immunoblotting, bull sperm were found to have a protein of molecular weight similar to mouse GalT at approximately 60 kDa. Immunogold low voltage scanning electron microscopy reveals that GalT epitopes are confined to the anterior cap of fresh or capacitated bull sperm. To investigate the function of bovine sperm GalT, fresh bull sperm were pretreated with either preimmune or anti-GalT antibody and added to in vitro-matured bovine oocytes. Sperm exposed to preimmune serum fertilized 82.7% (153 of 185) of the oocytes, whereas sperm exposed to anti-GalT antiserum fertilized only 42.3% (202 of 478) of the oocytes. We determined whether the inhibition of fertilization resulted from a direct inhibition of sperm-oocyte binding. The number of sperm bound to eggs was determined by low voltage scanning electron microscopy following pretreatment with preimmune or anti-GalT antibody. An average of 25.3+/-2.2 (mean +/- SEM) sperm bound per half-oocyte when treated with preimmune serum. In contrast, exposure of sperm to anti-GalT antiserum significantly lowered (P<0.001) the frequency of sperm binding to 9.9+/-0.8 bound per half-oocyte. These results show that GalT is present on the anterior cap of the bovine sperm head, where it participates in fertilization by facilitating sperm-oocyte binding. The function of GalT in both the murine and bovine systems suggests that it may serve as a generalized gamete receptor in mammals.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis.

Mouse male meiotic cytokinesis was studied using immunofluorescent probes against various elements of cytokinetic apparatus and electron microscopy. In normal mice, some spermatocytes fail to undergo cytokinesis after meiotic I or II nuclear divisions, forming syncytial secondary spermatocytes and spermatids. Abnormal cytokinetic cells develop sparse and dispersed midzone spindles during the early stage. However, during late stages, single and compact midzone spindles are formed as in normal cells, but localize asymmetrically and attach to the cortex. Myosin and f-actin were observed in the midzone spindle and midbody regions of normally cleaving cells as well as in those cells that failed to develop a cytokinetic furrow, implying that cytokinetic failure is unlikely to be due to defect in myosin or actin assembly. Depolymerization of microtubules by nocodazole resulted in the loss of the midbody-associated f-actin and myosin. These observations suggest that actin-myosin localization in the midbody could be a microtubule-dependent process that may not play a direct role in cytokinetic furrowing. Anti-centrin antibody labels the putative centrioles while anti-(gamma)-tubulin antibody labels the minus-ends of the midzone spindles of late-stage normal and abnormal cytokinetic cells, suggesting that the centrosome and midzone spindle nucleation in abnormal cytokinetic cells is not different from those of normally cleaving cells. Possible use of mouse male meiotic cells as a model system to study cytokinesis has been discussed.