Mysteries and unsolved problems of mammalian fertilization and related topics.

Mammalian fertilization is a fascinating process that leads to the formation of a new individual. Eggs and sperm are complex cells that must meet at the appropriate time and position within the female reproductive tract for successful fertilization. I have been studying various aspects of mammalian fertilization over 60 years.

Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules.

The present study was conducted to determine exact location where the acrosome reaction of fertilizing spermatozoa begins in the oviduct of the Chinese hamster. Unlike spermatozoa of other rodent species, Chinese hamster spermatozoa did not spontaneously undergo the acrosome reaction in fertilization-supporting media. In naturally mated females, spermatozoa in the uterus had intact acrosomes, whereas those in the lower oviductal isthmus had visibly thin acrosomal caps.

Ninety babies born after round spermatid injection into oocytes: survey of their development from fertilization to 2 years of age.

Objective: To compare physical and cognitive development of babies born after round spermatid injection (ROSI) with those born after natural conception.

Design: Comparison of efficiencies of ROSI and ICSI using testicular spermatozoa, performed in the St. Mother Clinic.

Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos.

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos.

Triathlon Medical Coverage: A Guide for Medical Directors.

Interest and participation in triathlon has grown rapidly over the past 20 yr and with this growth, there has been an increase in the number of new events. To maximize the safety of participation, triathlons require medical directors to plan and oversee medical care associated with event participation. Provision of proper medical care requires knowledge of staffing requirements, common triathlon medical conditions, impact of course design, communication skill, and a familiarity of administrative requirements.

The Behavior and Acrosomal Status of Mouse Spermatozoa In Vitro, and Within the Oviduct During Fertilization after Natural Mating.

Although 90%-100% of mouse oocytes can be fertilized in vitro with capacitated spermatozoa within 1 h after insemination, oocytes within the oviduct are fertilized one by one over a period of several hours. In vitro experiments showed that both acrosome-intact and acrosome-reacted spermatozoa entered the cumulus oophorus, but that acrosome-reacted spermatozoa reached the surface of oocytes more readily than acrosome-intact spermatozoa. During the period of fertilization within the oviduct, acrosome-reacted spermatozoa were seen throughout the isthmus, but with higher incidence in the upper than in the mid- and lower segments of the isthmus.

Fourteen babies born after round spermatid injection into human oocytes.

During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI).

Spata6 is required for normal assembly of the sperm connecting piece and tight head-tail conjunction.

"Pinhead sperm," or "acephalic sperm," a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis.

Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division.

Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred.

Sperm attractant in the micropyle region of fish and insect eggs.

In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal-a micropyle-through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance.

Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain.

Preimplantation genetic diagnosis is considered highly successful with respect to its accuracy in detecting genetic anomalies, although the effects of embryo biopsy on embryonic and fetal growth and development are less known, particularly in conjunction with IVF. Here, we compared biopsied (B) and nonbiopsied (NB) mouse embryos for developmental competence. Embryos C57BL/6 (B6) and B6D2F2 (F2) generated by IVF were subjected to single blastomere biopsy at the four-cell stage, and were either cultured for 120 h and subjected to differential inner cell mass (ICM) and trophoblast staining, or were transferred into the uterine tubes of surrogate mothers after 72 h of culture, to examine their pre- and postimplantation development, respectively.

Fertilization studies and assisted fertilization in mammals: their development and future.

Studies of mammalian fertilization progressed very slowly in the beginning because of difficulties in obtaining a large quantity of fully mature eggs at one time. With progression of techniques to collect and handle eggs and spermatozoa, research in mammalian fertilization advanced rapidly. Today, far more papers are published on mammalian gametes and fertilization than those of all other animals combined.

Problems of sperm fertility: a reproductive biologist's view.

For natural fertilization to occur successfully, millions of spermatozoa must be deposited in the lower end of the female genital tract during mating. Considerably fewer spermatozoa are required for fertilization when spermatozoa are deposited in the upper region of the female tract. The extreme case of assisted fertilization is the direct injection of a single spermatozoon into an oocyte in vitro, which is referred to as intracytoplasmic sperm injection or ICSI.

Germ cell research: a personal perspective.

My interest in germ cells began when I first witnessed sea urchin fertilization and embryo development during a laboratory class at Hokkaido University, Sapporo, Japan, almost 60 yr ago. Weismann's concept of germ cells that I learned during my undergraduate years became the driving force of my entire research career. During the early years, my associates and I used mainly the golden hamster and the guinea pig as model animals because their spermatozoa had large acrosomes and we could readily follow changes in the acrosomes without killing or staining spermatozoa.

A novel mechanism of late gene silencing drives SV40 transformation of human mesothelial cells.

Suppression of the late gene expression, usually by integration of the viral DNA into the host genome, is a critical step in DNA tumor virus carcinogenesis. SV40 induces high rates of transformation in infected primary human mesothelial cells in tissue culture, leading to the formation of immortal cell lines (SV40-transformed human mesothelial cell lines, S-HML). The studies described here were designed to elucidate the unusual susceptibility of primary human mesothelial cells to SV40 carcinogenesis.

Genomic DNA damage in mouse transgenesis.

Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage.

Ejaculated and epididymal mouse spermatozoa are different in their susceptibility to nuclease-dependent DNA damage and in their nuclease activity.

Ejaculated mouse sperm retrieved from the uteri are more susceptible to DNA damage during freeze-drying and freezing without cryoprotection than epididymal sperm. This prompted us to speculate that a factor present in the uterus after mating, either male or female derived, was responsible for increased susceptibility of ejaculated sperm to DNA damage during preservation and that the differences between epididymal and ejaculated mouse sperm in response to stress originated from varying nuclease activity. We first exposed epididymal sperm to the uterine content from females mated to vasectomized males (UCSP), to the uterine content from unmated females in estrus (UC), and to the seminal vesicle fluid (SVF) and examined sperm chromosomes after intracytoplasmic sperm injection (ICSI).

Preservation of ejaculated mouse spermatozoa from fertile C57BL/6 and infertile Hook1/Hook1 mice collected from the uteri of mated females.

Methods routinely used to preserve mouse spermatozoa require that the male be killed to recover spermatozoa from the epididymides. Here we obtained multiple samples of ejaculated spermatozoa from normal fertile C57BL/6 and infertile Hook1/Hook1 (formerly known as azh/azh) mutant males from uteri after mating, thus avoiding termination of the males. Ejaculated sperm were preserved by conventional cryopreservation or by rapid freezing without cryoprotection, and were injected into the oocytes by intracytoplasmic sperm injection (ICSI).

Male gamete contributions to the embryo.

During normal fertilization, plasma membranes of a spermatozoon and an oocyte mingle to form a mosaic plasma membrane of a zygote. This may contribute to the polyspermy block of the zygote. Sperm tail components (mitochondria, axonema, and accessory fibers) that enter the oocyte are "digested" without playing major roles in embryo development.

Comparison of oocyte-activating agents for mouse cloning.

Since somatic cell components are unable to activate oocytes following injection or fusion, enucleated oocytes receiving adult somatic cells during the cloning process must be activated artificially for their development. We compared the efficiency of four types of oocyte-activating agents: strontium, ethanol, single electric pulse, and spermatozoa. Although strontium was the best in supporting preimplantation development of reconstructed mouse oocytes, there was no significant difference among the four agents with respect to the rate of postimplantation embryo development and the birth of live offspring.

Fertilization and developmental initiation of oocytes by injection of spermatozoa and pre-spermatozoal cells.

The essence of fertilization is the union and mingling of male and female genomes. Therefore it is not surprising that microsurgical deposition of a single spermatozoon in an oocyte (ICSI) results in the development of normal offspring. Poorly motile or structurally aberrant spermatozoa, which are unable to fertilize under ordinary conditions, are not necessarily genomically abnormal.

Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI.

Background: Recent studies showed that ICSI with cauda epididymal or ejaculated sperm of infertile mice or men, respectively, was less effective in fertilization and normal embryo development than ICSI using sperm from the testes. These studies suggested that sperm nuclear quality declined after release from the testis, but the site where this loss of fertility occurs has not been localized.

Methods: We performed ICSI with testicular, caput, and cauda epididymal sperm from infertile Tnp1-/-Tnp2+/- mutant mice, which have a minimal level of transition nuclear proteins and are sterile by natural mating.