Yasuyuki Araki

In Vitro Murine Spermatogenesis in an Organ Culture System

Achieving mammalian spermatogenesis in vitro has a long history of research but remains elusive. The organ culture method has advantages over the cell culture method, because germ cells are in situ albeit the tissue as a whole is in vitro. The method was used in the 1960s and 1970s but encountered difficulties in inducing complete meiosis, i.e., in getting meiosis to proceed beyond the pachytene stage. In the present study, we reevaluated the organ culture method using two lines of transgenic mice, Acr-GFP and Gsg2 (haspin)-GFP mice, whose germ cells express green fluorescent protein (GFP) at the mid and end stages of meiosis onward, respectively. Immature testicular tissues from these mice, ranging from 4.5 to 14.5 days postpartum, were cultured on the surface of the medium, providing a liquid-gas interface. Culturing testicular tissues of all ages tested resulted in the expression of both Acr- and Gsg2-GFP. Round spermatids were identified by a combination of Gsg2-GFP expression, cell size, and the presence of a single nucleus with a dot stained by Hoechst. In addition, the chromosome number of one of such presumptive spermatids was found to be 20 by the premature chromosome condensation method. As our semiquantitative assay system using GFP expression grading was useful for monitoring the effects of different environmental factors, including temperature, oxygen concentration, and antiretinoic molecules, further improvement of the culture conditions should be possible in the future.

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.

Proliferation of Mouse Spermatogonial Stem Cells in Microdrop Culture

It is now possible to make mouse spermatogonial stem cells (SSCs) proliferate in vitro. However, these cultured cells, called germ-line stem (GS) cells, consist of not only SSCs but also a greater number of progenitor spermatogonia. Moreover, isolated GS cells barely proliferate. To elucidate the nature of SSCs and progenitor spermatogonia, we adapted a microdrop culture system to GS cells. Using a micromanipulator, individual microdrops were seeded with clusters or dissociated known numbers of GS cells. The number of surviving colonies was determined after 30 days. The proliferation rate of GS cells in microdrops increased as the number of GS cells seeded increased. It was observed that as few as three GS cells seeded in a microdrop can proliferate and expand the colony size. Those GS cells of expanded colonies were able to proliferate following subculture and underwent spermatogenesis in the host testis after transplantation into the seminiferous tubules of recipient mice. These data revealed that SSCs can multiply in a microdrop culture system. Microdrop culture offers a novel tool to elucidate the nature of SSCs in regard to their self-renewing capacity and can serve as a monitoring system of culture conditions for the self-renewal of SSCs.

Single human sperm cryopreservation method using hollow-core agarose capsules.

OBJECTIVE To develop an efficient cryopreservation method using a single sperm. DESIGN Experimental study. SETTING Laboratory of a private institute. PATIENT(S) A fertile donor. INTERVENTION(S) We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. MAIN OUTCOME MEASURE(S) The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. RESULT(S) The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. CONCLUSION(S) A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.

Bilateral tubal pregnancies after a single-embryo transfer

To present an extremely rare case of bilateral tubal pregnancies following a single‐embryo transfer in a woman with a 4 year history of infertility prior to seeking assisted reproductive technology.

Effects of cyclophosphamide administration on the in vitro fertilization of mice

To evaluate the oocyte fertilization ability and embryo growth after cyclophosphamide (CPA) treatment in mice.

Agarose capsules as new tools for protecting denuded mouse oocytes/embryos during handling and freezing-thawing and supporting embryonic development in vivo

Oocytes without a zona pellucida (ZP) often occur as a result of congenital or operational effects, but they are difficult to handle, and embryonic survival is low. Such zona-free (ZF) oocytes are therefore not used in clinics or laboratories. Furthermore, in the laboratory, removal of the ZP is often necessary for genetic manipulation by viral infection or twin production by blastomere separation, but adverse effects on development have been reported. It would therefore be extremely valuable if the embryo could be covered with a structure similar to that of the ZP. In this study, we sought to determine whether an agarose capsule could serve as a substitute for the ZP. Our results indicate that embryos derived from these agarose capsules were able to develop normally, and could be transplanted to obtain viable offspring, without having to remove the agarose capsule. Furthermore, before compaction, the agarose capsule embryos exhibited good freezing tolerance, and survival rate was extremely high compared to ZF embryos. Thus, agarose capsules represent a valuable tool for utilizing oocytes and embryos that lack a ZP, both in a clinical and livestock setting.