Steps of spermiogenesis in the ostrich (Struthio camelus).

Few studies describe the sequence of morphological events that characterize spermiogenesis in birds. In this paper, the clearly observable steps of spermiogenesis are described and illustrated for the first time in a commercially important ratite, the ostrich, based on light microscopy of toluidine blue-stained plastic sections. Findings were supplemented and supported by ultrastructural observations, PNA labeling of acrosome development, and immunocytochemical labeling of isolated spermatogenic cells. Spermiogenesis in the ostrich followed the general pattern described in non-passerine birds. Eight steps were identified based on changes in nuclear shape and contents, positioning of the centriolar complex, and acrosome development. Only two steps could be recognized with certainty during development of the round spermatid which contributed to the fewer steps recorded for the ostrich compared to that described in some other bird species. The only lectin that displayed acrosome reactivity was PNA and only for the first three steps of spermiogenesis. This suggests that organizational and/or compositional changes may occur in the acrosome during development and merits further investigation. Immunological labeling provided additional evidence to support the finding of previous studies that the tip of the nucleus in the ostrich is shaped by the forming acrosome and not by the microtubular manchette. To our knowledge, this is the first complete description of spermiogenesis in ostrich and one of few in any avian species. In addition to comparative reproduction and animal science, this work has implications for evolutionary biology as the reported germ cell features provide a bridge between reptile and ratite-avian spermatogenesis.

New Approaches to Boar Semen Evaluation, Processing and Improvement.

The improvement of boar reproductive performance may be the next frontier in reproductive management of swine herd in Unites States, facilitated by better understanding of boar sperm function and by the introduction of new advanced instrumentation in the andrology field. Objective single ejaculate evaluation and individual boar fertility prediction may be possible by introducing automated flow cytometric semen analysis with vital stains (e.g. acrosomal integrity and mito-potential), DNA fragmentation analysis and biomarkers (ubiquitin, PAWP, ALOX15, aggresome) associated with normal or defective sperm phenotypes. Measurement of sperm-produced reactive oxygen species (ROS) is a helpful indicator of normal semen sample. Semen ROS levels could be managed by the addition of ROS-scavenging antioxidants. Alternative energy regeneration substrates and sperm stimulants such as inorganic pyrophosphate and caffeine could increase sperm lifespan in extended semen and within the female reproductive system. Such technology could be combined with timed sperm release in the female reproductive system after artificial insemination. Sperm phenotype analysis by the image-based flow cytometry will go hand in hand with the advancement of swine genomics, linking aberrant sperm phenotype to the fertility influencing gene polymorphisms. Finally, poor-quality ejaculates could be rescued and acceptable ejaculates improved by semen purification methods such as the nanoparticle-based semen purification and magnetic-activated sperm sorting. Altogether, these scientific and technological advances could benefit swine industry, provided that the challenges of new technology adoption, dissemination and cost reduction are met.

Polo-like kinase 1 is essential for the first mitotic division in the mouse embryo.

Polo-like kinase 1 (PLK1), a member of the serine/threonine protein kinases family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little is known about PLK1 activities in the mammalian preimplantation embryo. We examined the role of PLK1 in the one-cell mouse embryo. Western blotting showed that the PLK1 protein content increased significantly during the S-phase of the one-cell stage and declined during the first mitotic division. Activation of PLK1 preceded nuclear envelope breakdown (NEBD) in both pronuclei at the entry to first embryo mitosis. Immunofluorescence revealed the presence of phosphorylated, active PLK1 (pThr(210) -PLK1) in both male and female pronuclei, and in the microtubule-organizing centers (MTOCs) shortly before NEBD. During the first mitotic metaphase, pThr(210) -PLK1 accumulated at the spindle poles and was also associated with condensed chromosomes. Inhibition of PLK1 activity with a specific PLK1 inhibitor, BI 2536, at the one-cell stage induced the formation of a bipolar spindle that displayed disordered microtubular arrangements and dislocated, condensed chromosomes. Although such embryos entered mitosis, they did not complete mitosis and arrested at metaphase. Time-lapse recording revealed progressive misalignment of condensed chromosomes during first mitotic metaphase. These data indicate that PLK1 activity is not essential for entry into first mitosis, but is required for the events leading up to metaphase-anaphase transition in the one-cell mouse embryo.

Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans.

Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS-PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.

Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction.

The objective of this study was to determine if a relationship exists between sperm parameters, measured by sperm chromatin structure assay (SCSA), and spontaneous abortion and multiple births in couples undergoing assisted reproduction treatment. Retrospective analysis of infertility treatment outcomes and occurrence of spontaneous abortion and multiple births was conducted in 233 couples who underwent treatment by intracytoplasmic sperm injection or intrauterine insemination at the Sher Institute for Reproductive Medicine, between 2001 and 2004. Sperm samples used for treatments were analysed for sperm concentration, sperm motility and two different parameters of SCSA (DNA fragmentation index, DFI, and high DNA stainability, HDS). Pregnancy, spontaneous abortion and multiple birth rates were recorded for all couples. A statistically significant relationship (P<0.001) was observed between DFI and spontaneous abortion. However, the correlation between HDS and spontaneous abortion was not statistically significant. Significantly lower levels of DFI were observed in men from couples having triplet pregnancies compared with those in the spontaneous abortion group (P ≤ 0.05). It is concluded that the parameters of SCSA correlate significantly with spontaneous abortion and multiple birth and may provide guidance for clinical decision making (number of embryos per transfer) and management of spontaneous abortion-prone cases.

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Timed artificial insemination plus heat II: gonadorelin injection in cows with low estrus expression scores increased pregnancy in progesterone/estradiol-based protocol.

The use of tail chalk and estrus/heat expression scores (HEATSC) evaluation is instrumental in identifying cows with greater estrus expression and greater artificial insemination pregnancy rates (P/AI) in cows submitted to timed artificial insemination (TAI), and cows with low or no estrus expression present lower P/AI. It was intended in this study to improve the pregnancy rates in TAI for Bos indicus beef cows, and gonadotrophin-releasing hormone (GnRH) injection was hypothesized to increase pregnancy rates in a TAI program for cows submitted to progesterone-estradiol-based protocols with low or no estrus expression, evaluated by HEATSC. Cows (n= 2284) received a progesterone device and 2 mg estradiol benzoate, after 8 days the device was removed and 1 mg estradiol cypionate, 150 µg of d-cloprostenol and 300 IU equine chorionic gonadotropin was administered. All cows were marked with chalk and HEATSC evaluated (scales 1 to 3) at TAI performed on day 10. Animals with HEATSC1 and HEATSC2 (n= 937) received 100 µg de gonadorelin (GNRH group; n= 470), or 1 ml saline (Control group; n= 467), and cows with HEATSC3 (named HEAT group; n= 1347) received no additional treatment. The larger dominant follicle, evaluated on day 8and at TAI (day 10), was greater in HEAT group (P= 0.0145 and P <0.001, respectively). Corpus luteum (CL) area and progesterone concentration was evaluated on day 17, and CL area was larger in HEAT group, intermediary in Control and lower in GnRH group (Control= 2.68 cm2, GnRH= 2.37 cm2, HEAT group= 3.07 cm2, P <0.001). Greater progesterone concentrations were found in HEAT group than in Control and GnRH groups (Control= 4.74 ng/ml, GnRH= 4.29 ng/ml, HEAT group= 6.08 ng/ml, P<0.001). There was a difference in ovulation rate, greater in HEAT group than GnRH and Control groups (Control= 72.5%; GnRH= 81.25%; HEAT group= 90.71%; P= 0.0024). Artificial insemination pregnancy rates was greater in HEAT group (57.09% (769/1347) than in Control and GNRH groups, with positive effect of GnRH injection at the time of TAI in P/AI (Control= 36.18% (169/467), GnRH= 45.95% (216/470); P<0.0001). In conclusion, GnRH application in cows with low HEATSC (1 and 2) is a simple strategy, requiring no changes in TAI management to increase pregnancy rates in postpartum beef cows submitted to progesterone-estradiol-based TAI protocols, without reaching, however, the pregnancy rates of cows that demonstrate high estrus expression at the TAI.

Timed artificial insemination plus heat I: effect of estrus expression scores on pregnancy of cows subjected to progesterone-estradiol-based protocols.

Expression of estrus near timed artificial insemination (TAI) is associated with greater fertility, and estrus detection could improve TAI fertility or direct TAI management, although accurate estrus detection can be difficult and time-consuming using traditional methods. The aim of this study is to evaluate influence of estrus on pregnancy (artificial insemination pregnancy rates (P/AI)) and to validate an alternative method to classify estrus/heat expression using tail chalking (HEATSC) in postpartum Bos indicus cows subjected to TAI in progesterone-estrogen-based protocols. In experiment 1 (Exp. 1), cows (5491) were subjected to visual observation of estrus after progesterone device removal, before TAI, and P/AI was evaluated according to estrus and body condition score (BCS). Cows received a progesterone device and 2 mg estradiol benzoate (EB). After 8 days, the device was removed and 150 µg of d-cloprostenol and 300 IU equine chorionic gonadotrophin was given. Later, animals in Exp. 1 received 1 mg EB and TAI 44 to 48 h. In the Exp. 2 - 3830 cows using similar protocol, received different ovulation inducers: 1 mg EB (n=1624) or 1 mg estradiol cypionate (EC; n=2206) on day 8 (D8). Cows were then marked with chalk, and HEATSC evaluated at TAI on D10 (HEATSC1 - no chalk removal=no estrus expression; HEATSC2 - partial chalk removal=low estrus expression; HEATSC3 - near complete/complete chalk removal=high estrus expression). In Exp. 1, cows showing estrus presented greater P/AI (48.4% v. 40.2%, P<0.05). In Exp. 2, P/AI (HEATSC1 - 40.0%; HEATSC2 - 49.7%; HEATSC3 - 60.9%; P<0.001), and larger follicle timed artificial insemination (LFTAI) (<0.001) varied according to HEATSC. There was no difference in P/AI (P=0.41) or LFTAI (P=0.33) according to ovulation inducer. Cows with greater BCS showed greater P/AI in both experiments (P<0.05). Estrus presence and greater HEATSC improved P/AI, and EC v. EB used promoted differential estrus manifestation (cows showing HEATSC2 and HEATSC3: 79.5% with EB v. 69.98% with EC use, P<0.001), however, with similar P/AI. The use of HEATSC in B. indicus cows subjected to TAI is useful to identify cows with greater estrus expression and consequently improved pregnancy rates in TAI, allowing the cows with low HEATSC to be targeted for additional treatments aimed at improving P/AI.

Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development.

Mitochondrial transcription factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA). We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig (Sus scrofa). We hypothesized that TFAM is not present in the boar sperm mitochondria to reduce the possibility of paternal mtDNA propagation in the progeny. In contrast, we anticipated that Tfam gene is expressed in a developmental stage-dependent manner in porcine oocytes and embryos. The appropriate TFAM band of 25 kDa was detected by Western blotting in ejaculated boar spermatozoa, as well as in porcine oocytes and zygotes. Boar sperm extracts also displayed several bands >25 kDa suggestive of post-translational modification by ubiquitination, confirmed by affinity purification of ubiquitinated proteins. TFAM immunoreactivity was relegated to the sperm tail principal piece and sperm head in fully differentiated spermatozoa. The content of Tfam mRNA increased considerably from the germinal vesicle to blastocyst stage and also between in vitro fertilized and cultured blastocysts compared to in vivo-derived blastocysts. TFAM protein accumulated in the oocytes during maturation and was reduced by proteolysis after fertilization. This pattern was not mirrored in parthenogenetically activated oocytes and zygotes reconstructed by SCNT, suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. Thus, TFAM may exert a critical role in porcine gametogenesis and preimplantation embryo development. Altogether, our data on the role of TFAM in mitochondrial function and inheritance have broad implications for cell physiology and evolutionary biology.

Review: Sperm-oocyte interactions and their implications for bull fertility, with emphasis on the ubiquitin-proteasome system.

Fertilization is an intricate cascade of events that irreversibly alter the participating male and female gamete and ultimately lead to the union of paternal and maternal genomes in the zygote. Fertilization starts with sperm capacitation within the oviductal sperm reservoir, followed by gamete recognition, sperm-zona pellucida interactions and sperm-oolemma adhesion and fusion, followed by sperm incorporation, oocyte activation, pronuclear development and embryo cleavage. At fertilization, bull spermatozoon loses its acrosome and plasma membrane components and contributes chromosomes, centriole, perinuclear theca proteins and regulatory RNAs to the zygote. While also incorporated in oocyte cytoplasm, structures of the sperm tail, including mitochondrial sheath, axoneme, fibrous sheath and outer dense fibers are degraded and recycled. The ability of some of these sperm contributed components to give rise to functional zygotic structures and properly induce embryonic development may vary between bulls, bearing on their reproductive performance, and on the fitness, health, fertility and production traits of their offspring. Proper functioning, recycling and remodeling of gamete structures at fertilization is aided by the ubiquitin-proteasome system (UPS), the universal substrate-specific protein recycling pathway present in bovine and other mammalian oocytes and spermatozoa. This review is focused on the aspects of UPS relevant to bovine fertilization and bull fertility.

Relative levels of semen platelet activating factor-receptor (PAFr) and ubiquitin in yearling bulls with high content of semen white blood cells: implications for breeding soundness evaluation.

High content of the platelet activating factor (PAF) and its plasma membrane receptor (PAFr) in semen is thought to benefit fertility in farm animals and humans. We used flow cytometric, biochemical, and immunocytochemical analysis to examine PAFr levels alone (Trial 1, n = 156 bulls) or in a dual assay with sperm defect marker ubiquitin (UBI; Trial 2, n = 88 bulls), in semen samples from 160 yearling bulls undergoing Breeding Soundness Evaluations (BSE). In both trials, we observed increased PAFr levels in semen samples with high content of white blood cells (WBC). Consequently, PAFr levels within such semen samples correlated negatively with several subjective parameters of BSE, including palpation, satisfaction of evaluation, and scrotal circumference. Due to a high WBC content, increased semen sample dilution had to be applied for microscopic evaluation. There was a negative correlation between semen PAFr and conventional sperm morphology, while the increased levels of PAFr correlated positively with sperm UBI content. Immunofluorescence microcopy revealed high expression of PAFr on the surface of leukocytes and morphologically normal spermatozoa, while reduced immunoreactivity was observed in defective spermatozoa immunoreactive to anti-UBI antibodies. A single PAFr band of appropriate mass was observed in Western blots of ejaculated spermatozoa, while testicular and epididymal spermatozoa also displayed several larger bands indicative of posttranslational processing or modification. Collectively, these data suggest that high levels of semen PAFr in young bulls are indicative of semen contamination with WBC. In the future, objective protein marker-based semen analyses in young bulls will likely require additional parameters distinguishing between marker expression in the spermatozoa and in the contaminating WBC. While identification of high sperm PAFr levels may support fertility, this assay alone is not reliable, due to the expression of PAFr in WBC that contaminate semen samples.

Arachidonate 15-lipoxygenase and ubiquitin as fertility markers in boars.

Accurate semen analysis is an important issue in the swine industry. We evaluated two candidate fertility marker proteins associated with sperm cytoplasmic droplet (CD), including 15-lipoxygenase (15-LOX) and ubiquitin (UBI) in a controlled single-sire artificial insemination (AI) trial. Ejaculates (n=116) were collected from 18 fertile Large White boars monthly for 8 mo, and analyzed by semi-quantitative, densitometry-based Western blotting and flow cytometry with antibodies against 15-LOX and UBI. Data were correlated with farrowing rates (FR) and total numbers of piglets born (TNB) from 1754 AI services by 13 of 18 boars, and compared with a conventional microscopic semen analysis. In semi-quantitative Western blotting, both 15-LOX and UBI were correlated with seasonal changes in the percentage of normal (r=-0.38, P<0.01; r=-0.27, P<0.05, respectively) and CD-bearing spermatozoa (r=0.35, P<0.01; r=0.27, P<0.05, respectively). In flow cytometry, UBI and 15-LOX levels showed seasonal changes coinciding with seasonal changes of FR and TNB, representing 13 boars, 88 ejaculates and 1,232 AI services. There were correlations between flow cytometric values of UBI and FR (r=0.31; P<0.05), adjusted FR (r=0.30; P<0.05), TNB (r=-0.38; P<0.01) and adjusted TNB (r=-0.37; P<0.01). Flow cytometric measurements of 15-LOX correlated negatively with TNB (r=-0.33; P<0.05) and adjusted TNB (r=-0.34; P<0.05). These data suggested that boar fertility estimation could be achieved within a group of fertile boars by the use of objectively measurable fertility markers. Flow cytometry appeared more informative and more practical than semi-quantitative Western blotting. This technology could be further optimized for the selection of the most fertile sires in an artificial insemination program.

Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents gamma-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. gamma-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal gamma-tubulin. Recruitment of maternal gamma-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Paternal contributions to the mammalian zygote: fertilization after sperm-egg fusion.

Mammalian fertilization has traditionally been regarded as a simple blending of two gametes, during which the haploid genome of the fertilizing spermatozoon constitutes the primary paternal contribution to the resulting embryo. In contrast to this view, new research provides evidence of important cytoplasmic contributions made by the fertilizing spermatozoon to the zygotic makeup, to the organization of preimplantation development, and even reproductive success of new forms of assisted fertilization. The central role of the sperm-contributed centriole in the reconstitution of zygotic centrosome has been established in most mammalian species and is put in contrast with strictly maternal centrosomal inheritance in rodents. The complementary reduction or multiplication of sperm and oocyte organelles during gametogenesis, exemplified by the differences in the biogenesis of centrosome in sperm and oocytes, represents an intriguing mechanism for avoiding their redundancy during early embryogenesis. New studies on perinuclear theca of sperm revealed its importance for both spermatogenesis and fertilization. Remodeling of the sperm chromatin into a male pronucleus is guided by oocyte-produced, reducing peptide glutathione and a number of molecules required for the reconstitution of the functional nuclear envelope and nuclear skeleton. Although some of the sperm structures are transformed into zygotic components, the elimination of others is vital to early stages of embryonic development. Sperm mitochondria, carrying potentially harmful paternal mtDNA, appear to be eliminated by a ubiquitin-dependent mechanism. Other accessory structures of the sperm axoneme, including fibrous sheath, microtubule doublets, outer dense fibers, and the striated columns of connecting piece, are discarded in an orderly fashion. The new methods of assisted fertilization, represented by intracytoplasmic sperm injection and round spermatid injection, bypass multiple steps of natural fertilization by introducing an intact spermatozoon or spermatogenic cell into oocyte cytoplasm. Consequently, the carryover of sperm accessory structures that would normally be eliminated before or during the entry of sperm into oocyte cytoplasm persist therein and may interfere with early embryonic development, thus decreasing the success rate of assisted fertilization and possibly causing severe embryonic anomalies. Similarly, foreign organelles, proteins, messenger RNAs, and mitochondrial DNAs, which may have a profound impact on the embryonic development, are propagated by the nuclear transfer of embryonic blastomeres and somatic cell nuclei. This aspect of assisted fertilization is yet to be explored by a focused effort.

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.

Atypical decondensation of the sperm nucleus, delayed replication of the male genome, and sex chromosome positioning following intracytoplasmic human sperm injection (ICSI) into golden hamster eggs: does ICSI itself introduce chromosomal anomalies?

OBJECTIVE: To examine nuclear decondensation, positioning of sex chromosomes, and the S-phase in human sperm nuclei following intracytoplasmic sperm injection (ICSI) into hamster eggs. DESIGN: Prospective analysis of hamster eggs and human sperm following ICSI. SETTING: Division of Reproductive Sciences, Oregon Health Sciences University and Oregon Regional Primate Research Center. PATIENT(S): Fertile donor sperm from a commercial source. INTERVENTION(S): Human sperm were examined by immunofluorescence stain, bromodioxyuridine (BrdU) uptake assay and fluorescence in situ hybridization following ICSI into hamster eggs. MAIN OUTCOME MEASURE(S): Immunostaining and fluorescence in situ hybridization. RESULT(S): Decondensation of human sperm nuclei occurred initially in the basal region, and perinuclear theca of sperm persisted around the condensed apical region. In some sperm nuclei, following ICSI the sex chromosomes were in the apical region, remaining condensed for longer than in the basal region. S-phase entry of human sperm nuclei following ICSI was delayed compared to the zona-free hamster egg penetration assay. CONCLUSION(S): These results force questions about the mechanism of male pronuclear formation after ICSI and suggest new strategies for understanding the basis of chromosomal anomalies leading to birth defects as well as continuing improvements in the safety and efficacy of infertility therapies.

Cell and molecular biological challenges of ICSI: ART before science?

Authors discuss the possible genetic and cell biological risks to offspring conceived by ICSI in relation to the lack of fundamental research using relevant animal models.

Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes.

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.

Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.

The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.