Vanesa Y. Rawe

Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization

Fertilization is complete once the parental genomes unite, and requires the migration of the egg nucleus to the sperm nucleus (female and male pronuclei, respectively) on microtubules within the inseminated egg. Neither the molecular mechanism of pronucleus binding to microtubules nor the role of motor proteins in regulating pronuclear motility has been fully characterized, and the failure of zygotic development in some patients suggests that they contribute to human infertility. Based on the minus-end direction of female pronuclear migration, we propose a role for cytoplasmic dynein and dynactin in associating with the pronuclear envelope and mediating genomic union. Our results show that dynein intermediate and heavy chains preferentially concentrate around the female pronucleus, whereas dynactin subunits p150Glued, p50 and p62 localize to the surfaces of both pronuclei. Transfection of antibodies against dynein and dynactin block female pronuclear migration in zygotes. Both parthenogenetic activation in oocytes and microtubule depolymerization in zygotes significantly reduce the localization of dynein to the female pronucleus but do not inhibit the pronuclear association of dynactin. When immunoprecipitated from zygotes, p150Glued associates with nuclear pore complex proteins, as well as the intermediate filament vimentin and dynein. Antibodies against nucleoporins and vimentin inhibit pronuclear apposition when transfected into zygotes. We conclude that preferentially localized dynein and perinuclear dynactin associate with the nuclear pore complex and vimentin and are required to mediate genomic union. These data suggest a model in which dynein accumulates and binds to the female pronucleus on sperm aster microtubules, where it interacts with dynactin, nucleoporins and vimentin.

Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI.

Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.

Semen Levels of Spermatid-Specific Thioredoxin-3 Correlate with Pregnancy Rates in ART Couples

Spermatid specific thioredoxin-3 (SPTRX3 or TXNDC8) is a testis/male germ line specific member of thioredoxin family that accumulates in the superfluous cytoplasm of defective human spermatozoa. We hypothesized that semen levels of SPTRX3 are reflective of treatment outcome in assisted reproductive therapy (ART) couples treated by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Relationship between SPTRX3 and treatment outcome was investigated in 239 couples undergoing ART at an infertility clinic. Sperm content of SPTRX3 was evaluated by flow cytometry and epifluorescence microscopy, and correlated with clinical semen analysis parameters, and data on embryo development and pregnancy establishment. High SPTRX3 levels (>15% SPTRX3-positive spermatozoa) were found in 51% of male infertility patients (n = 72), in 20% of men from couples with unexplained, idiopathic infertility (n = 61) and in 14% of men from couples previously diagnosed with female-only infertility (n = 85). Couples with high SPTRX3 produced fewer two-pronuclear zygotes and had a reduced pregnancy rate (19.2% pregnant with >15% SPTRX3-positive spermatozoa vs. 41.2% pregnant with <5% SPTRX3-positive sperm; one-sided p<0.05). The average pregnancy rate of all 239 couples was 25.1%. Live birth rate was 19.2% and lowest average SPTRX3 levels were found in couples that delivered twins. Men with >15% of SPTRX3-positive spermatozoa, a cutoff value established by ROC analysis, had their chance of fathering children by IVF or ICSI reduced by nearly two-thirds. The percentage of SPTRX3-positive spermatozoa had predictive value for pregnancy after ART. Gradient purification and sperm swim-up failed to remove all SPTRX3-positive spermatozoa from semen prepared for ART. In summary, the elevated semen content of SPTRX3 in men from ART couples coincided with reduced incidence of pregnancy by IVF or ICSI, identifying SPTRX3 as a candidate biomarker reflective of ART outcome.

Exploring the cytoskeleton during intracytoplasmic sperm injection in humans.

Understanding the cellular events during fertilization in mammals is a major challenge that can contribute to the improvement of future infertility treatments in humans and reproductive performance in farm animals. Of special interest is the role of the oocyte and sperm cytoskeleton during the initial interaction between gametes. The aim of this chapter is to describe methods for studying cytoskeletal features during in vitro fertilization after intracytoplasmic sperm injection (ICSI) in humans. The following protocols will provide a detailed description of how to perform immunodetection and imaging of human eggs, zygotes, and sperm by fluorescence (confocal and epifluorescence) and electron microscopy.

Biogenesis of the sperm head perinuclear theca during human spermiogenesis

We analyzed the appearance and localization of the sub-acrosomal perinuclear theca (PT) during human spermiogenesis. The PT is tightly associated with acrosomal biogenesis.

Microtubules and parental genome organisation during abnormal fertilisation in humans

We analysed the distribution of beta-tubulins, acetylated alpha-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, beta-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.

Pronuclear abnormalities and cytoskeletal organization during assisted fertilization in a patient with multifollicular ovarian response.

AbstractPurpose: To analyze the distribution of α tubulins and acetylated α tubulins and the chromatin configuration in abnormally fertilized zygotes from a patient with a multifollicular ovarian response after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Methods: Immunofluorescence and phase contrast microscopy was performed in abnormally fertilized zygotes. Results: After phase contrast microscopy analysis, immunofluorescence staining was performed in 20 oocytes that developed ≥3 pronuclei (PN) and karyomeres after IVF–ICSI. Around 80% of the abnormal zygotes from IVF were the consequence of monospermic fertilizations. Retention of the second polar body (PB) and the presumptive split of ≥1 PN within the cytoplasm were the main events present in most oocytes after IVF–ICSI. Conclusions: Fluorescence labeling of selected sperm and oocyte components affords a unique view of abnormal fertilized zygotes. Surprisingly, anomalies detected after IVF–ICSI showed similar etiologies in this special group of zygotes.

Correction: Semen Levels of Spermatid-Specific Thioredoxin-3 Correlate with Pregnancy Rates in ART Couples

Affiliation number 7 for the eighth author, Antonio Miranda-Vizuete, is incorrect. The correct affiliation is: Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Sevilla, Spain.