Mito Kanatsu-Shinohara

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F₁ offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F₁ offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation–ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F₂ generation. Furthermore, although these F₁ offspring appeared normal, F₂ offspring produced by IVF using F₁ sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.

Vast amounts of sperm are produced from spermatogonial stem cells (SSCs), which continuously undergo self-renewal. We examined the possible effect of laterality in male germline transmission efficiency of SSCs using a spermatogonial transplantation technique. We transplanted the same number of wild-type and Egfp transgenic SSCs in the same or different testes of individual recipient mice and compared the fertility of each type of recipient by natural mating. Transgenic mice were born within 3 months after transplantation regardless of the transplantation pattern. However, transgenic offspring were born at a significantly increased frequency when wild-type and transgenic SSCs were transplanted separately. In addition, this type of recipient sired significantly more litters that consisted exclusively of transgenic mice, which suggested that left and right testes have different time windows for fertilization. Thus, laterality plays an important role in germline transmission patterns from SSCs.

Significance: Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis. Previous studies on SSC self-renewal have focused on the positive regulation, but we hypothesized that negative regulation of self-renewal is equally important in understanding SSC self-renewal. We show here that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box–type ubiquitin ligase, negatively regulates SSC self-renewal. FBXW7 is expressed only in the undifferentiated spermatogonia compartment, and its overexpression compromises SSC activity whereasFbxw7deficiency enhances SSC colonization and caused accumulation of undifferentiated spermatogonia. We also demonstrate that FBXW7 regulates stability of myelocytomatosis oncogene (MYC), which increased SSC activity upon overexpression. These results suggest that FBXW7 counteracts with positive regulators of self-renewal by degrading MYC and demonstrate the importance of negative regulators of self-renewal in understanding SSC homeostasis.

Spermatogenesis originates from spermatogonial stem cells (SSCs). Development of the spermatogonial transplantation technique in 1994 provided the first functional assay to characterize SSCs. In 2000, glial cell line–derived neurotrophic factor was identified as a SSC self-renewal factor. This discovery not only provided a clue to understand SSC self-renewing mechanisms but also made it possible to derive germline stem (GS) cell cultures in 2003. In vitro culture of GS cells demonstrated their potential pluripotency and their utility in germline modification. However, in vivo SSC analyses have challenged the traditional concept of SSC self-renewal and have revealed their relationship with the microenvironment. An improved understanding of SSC self-renewal through functional assays promises to uncover fundamental principles of stem cell biology and will enable us to use these cells for applications in animal transgenesis and medicine.

Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.

Spermatogonial stem cells (SSCs) undergo self-renewal division to support spermatogenesis. Although several positive regulators of SSC self-renewal have been identified, little is known about the mechanisms that negatively regulate SSCs. Here we developed a novel transplantation assay for SSCs and demonstrate that p21 and p27 cyclin-dependent kinase inhibitors play critical roles in SSC self-renewal and differentiation. Overexpression of p21 or p27 abrogated proliferation of cultured SSCs in vitro, and their expression levels were downregulated by exogenous self-renewal signals. In contrast, no apparent defects were found in p21 or p27-deficient SSCs by spermatogonial transplantation. However, competitive spermatogonial transplantation with WT SSCs revealed that the loss of either gene causes distortion of germline transmission: p21-deficiency facilitated mutant offspring production, whereas germline transmission was limited by p27-deficiency. Serial transplantation also showed that the loss of p27, but not p21, decreases secondary colony formation, suggesting that appropriate amounts of p27 are necessary for sustaining SSC self-renewal. Thus, p21 and p27 cyclin-dependent kinase inhibitors play critical roles in germline transmission by regulating the balance between SSC self-renewal and differentiation, and competitive spermatogonial transplantation technique will be useful for analyzing subtle defects in spermatogenesis that are not evident by traditional spermatogonial transplantation.