Diagnosing cellular defects in an unexplained case of total fertilization failure.

Despite the advent of ICSI, cases of total fertilization failure (TFF) often lead to cycle cancellation with limited diagnostic and therapeutic strategies currently available. We report on the case of an infertile couple who failed to conceive after repeated IVF and ICSI. Sperm of the husband were morphologically normal and passed a functional test assessing their ability to activate mouse oocytes. Whether oocytes were activated artificially with calcium ionophore after injection of husband's or with donor sperm, all oocytes failed to fertilize. Multiple polar bodies and two disorganized spindle structures were predominantly observed, pointing towards a cytoplasmic defect in the oocytes as the primary cause of the couple's infertility. In fact, injection of husband's sperm into donor oocytes resulted in the delivery of healthy twins. This report describes a course of action that may be applied for couples with TFF after both IVF and ICSI.

Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei.

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.

Lack of Spem1 causes aberrant cytoplasm removal, sperm deformation, and male infertility.

We identified a previously uncharacterized gene, spermatid maturation 1 (Spem1), encoding a protein exclusively expressed in the cytoplasm of steps 14-16 elongated spermatids in the mouse testis. This protein contains no known functional domains and is highly conserved across mammalian species. Male mice deficient in Spem1 were completely infertile because of deformed sperm characterized by a bent head wrapped around by the neck and the middle piece of the tail. We show that lack of Spem1 causes failure of the cytoplasm to become loose and detach from the head and the neck region of the developing spermatozoa. Retained cytoplasmic components mechanically obstruct the straightening of the sperm head and the stretching of the growing tail, leading to the bending of the head in the neck, followed by the wrapping of the head by the neck or the middle piece of the sperm tail. Our study reveals that proper cytoplasm removal is a genetically regulated process requiring the participation of Spem1 and that lack of Spem1 causes sperm deformation and male infertility.

Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development.

Direct injection of a single spermatozoon into an oocyte (ICSI) can produce apparently normal offspring. Although the production of normal offspring by ICSI has been successful in mice and humans, it has been less successful in many other species. The reason for this is not clear, but could be, in part, due to inconsistent activation of oocytes because of delayed disintegration of sperm plasma membrane within oocytes and incorporation of the acrosome containing a spectrum of hydrolyzing enzymes. In the mouse, the removal of sperm plasma membrane and acrosome was not a prerequisite to produce offspring by ICSI, but it resulted in earlier onset of oocyte activation and better embryonic development. The best result was obtained when spermatozoa were demembranated individually immediately before ICSI by using lysolecithin, a hydrolysis product of membrane phospholipids.

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring.

Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. We tested whether mouse male germ cells could be cryopreserved without cryoprotection by simply freezing epididymides, testes, or whole bodies. The reproductive organs were isolated from killed mice and frozen for 1 week to 1 year at -80 degrees C before spermatozoa and spermatids were collected and injected into mature oocytes. Normal pups were born irrespective of strains tested (ICR and C57BL/6). Epididymides and testes frozen and transported internationally to another laboratory by air could produce pups of inbred C57BL/6 mice. Testicular spermatozoa retrieved from the bodies of male mice (BALB/c nude and C3H/He strains) that had been kept frozen (-20 degrees C) for 15 years could also produce normal offspring by microinsemination. Thus, freezing of either male reproductive organs or whole bodies is the simplest way to preserve male germ cells. Restoration of extinct species could be possible if male individuals are found in permafrost.

Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development.

In mice and humans, a normal offspring can be obtained by injecting a single spermatozoon into an oocyte, the process called intracytoplasmic sperm injection (ICSI). When three or more mouse spermatozoa with intact acrosomes were injected into individual mouse oocytes, an increasing proportion of oocytes became deformed and lysed. Oocytes did not deform and lyse when acrosome-less spermatozoa were injected, regardless of the number of spermatozoa injected. Injection of more than four human spermatozoa into a mouse oocyte produced vacuole-like structures in each oocyte. This vacuolation did not happen when spermatozoa were freed from acrosomes before injection. Hamsters, cattle, and pigs have much larger acrosomes than the mouse or human. Injection of a single acrosome-intact hamster, bovine, and porcine spermatozoon deformed and lysed many or all mouse oocytes. This deformation did not happen when these spermatozoa were freed from acrosomes before ICSI, regardless of the number of spermatozoa injected. Because trypsin and hyaluronidase mimicked the action of acrosome-intact spermatozoa, it is likely that the acrosomal enzymes deform and lyse the oocytes. Injection of small amounts of trypsin and hyaluronidase into normally fertilized mouse eggs disturbed their pre- and postimplantation development. In view of potentially harmful effects of acrosomal enzymes on embryo development, the removal of acrosomes before ICSI is recommended for animals with large sperm acrosomes. The removal of acrosomes may increase the efficiency of ICSI in these animals. Although human and mouse spermatozoa do not need to be freed from acrosomes, the removal of acrosomes before ICSI is theoretically preferable.

Fertilization failure from a sperm chromatin defect in couples with unexplained infertility.

OBJECTIVE: To investigate the relationship between unexplained infertility and fertilization failure from nucleoprotein defects in ejaculated human sperm and to study the usefulness of sperm chromatin assays, using AO fluorescence dye, to evaluate patients with unexplained infertility before treatment. STUDY DESIGN: From January 1999 to January 2000, 513 infertile couples had the clinical causes of their infertility assessed. During the next investigative period (February 2000-February 2001), 137 cases of unexplained infertility (n = 80) were chosen for this study, as were cases of tubal factor infertility (n = 57) as controls. The status of nuclear chromatin in ejaculated sperm was examined using acridine orange staining, followed by a conventional in vitro fertilization procedure. RESULTS: The number of patients with immature ejaculated sperm was 16 of 30 (53.3%) unexplained infertility cases involving fertilization failure, 8 of 50 (16.0%) unexplained infertility cases without fertilization failure and 5 of 57 (8.8%) tubal factor infertility cases. A significant difference was observed between unexplained infertility cases with fertilization failure and the other groups (P < .0001). CONCLUSION: These results suggest that the nuclear immaturity of ejaculated human sperm may be 1 of the primary factors underlying unexplained infertility.

Chromosomal analysis of mouse spermatozoa following physical and chemical treatments that are effective in inactivating HIV.

Human immunodeficiency virus (HIV) can be inactivated by heating at 56 degrees C for 30 min, treating with 50% ethanol at room temperature for 10 min, or treating with 2% sodium hypochlorite solution (NaClO) at room temperature for 60 min. Using a mouse model, we evaluated the risk of generating chromosome damage in spermatozoa following these treatments. The spermatozoa were all dead after the treatments. Although 41.3% of oocytes injected with ethanol-treated spermatozoa successfully activated, none of the oocytes injected with heated or NaClO-treated spermatozoa activated. When artificial stimulation with strontium was used, the fertilization of oocytes with heated or ethanol-treated spermatozoa was completely rescued. Sperm nuclei treated with NaClO neither decondensed nor developed to a male pronucleus. The incidences of structural chromosome aberrations in 1-cell zygotes derived from the heated spermatozoa (45.6%) and ethanol-treated spermatozoa (91.2%) were significantly higher than those in the matched controls (5.5% and 10.5%, respectively). Further study is needed to develop a methodology for the protection of spermatozoa against chromosome damage or the separation of damaged spermatozoa before intracytoplasmic sperm injection.