Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.I n most mammalian species, sex is determined by the presence or absence of the Y chromosome. In mice, the Sry gene locates to the minimum sex-determining region of the murine Y chromosome 1 , is expressed in the male genital ridge at the time of sex determination 2 and has been proven to be a sex-determining gene based on gain-of-function experiments, i.e., the overexpression of Sry in XX mice achieved with transgenic mouse technology reveals a male phenotype 3 . Also in humans, the SRY gene has been shown to play a pivotal role in sex determination: point mutations or deletions of the SRY gene are found in approximately 15% of XY females, and translocated SRY is found in the autosomes of most XX males 4 . Although there are a number of suggestive observations, it is important to confirm the function of Sry in vivo using loss-of-function analyses with targeted mutagenesis in order to examine whether Sry is the one and only sex-determining gene on the Y chromosome and to finally confirm the Sry gene as the sex-determining gene and provide an animal model of XY female syndrome. However, it is difficult to create knockout (KO) mice of Y-linked genes using conventional homologous recombination-based methods with embryonic stem (ES) cells, as the process requires an adequate length of specific sequences of homologous arms to construct a KO vector, and the Y chromosome is rich in repeats.In 2013, Sung et al. 5 first reported that KO mice can be produced using transcription activator-like effector nuclease (TALEN) technology without conventional homologous recombination-based methods. TALEN protein is an artificial sequence-specific endonuclease that contains Xanthomonas transcription activator-like effector (TALE) and a nuclease domain of FokI restriction endonuclease 6 . DNA binding domain of TALE consists of a tandem repeat of 33-35 amino acid motifs in which there are two critical adjacent amino acid pairs called a repeat variable diresidue (RVD) that determines the binding specificity for single nucleotide. There is a one-toone relationship between the RVD and its recognition nucleotide 7,8 . Using this code, a TALEN can...

Testosterone masculinizes the brain and bodily functions and promotes the development of male characteristics. In the brain, there is a critical period during which testosterone acts on neural circuits to stimulate the development of male-type neural characteristics.Early exposure to testosterone masculinizes reproductive, cognitive, emotional, and neurophysiological functions, such as stress responses (McCarthy & Arnold, 2011). In the developing mammalian brain, testosterone can either be metabolized to estrogen via aromatase, dihydrotestosterone (DHT) via 5α-reductase, or remain unmetabolized. Testosterone and DHT act through a single androgen receptor (AR), while estrogen acts through two types of estrogen receptors (ERα and ERβ) (McCarthy & Arnold, 2011). The relative

Testosterone masculinizes male sexual behavior through an organizational and activational effects. We previously reported that the emission of ultrasonic vocalizations (USVs) in male mice was dependent on the organizational effects of testosterone; females treated with testosterone in the perinatal and peripubertal periods, but not in adults, had increased USV emissions compared to males. Recently, it was revealed that male USVs have various acoustic characteristics and these variations were related to behavioral interactions with other mice. In this regard, the detailed acoustic characteristic changes induced by testosterone have not been fully elucidated. Here, we revealed that testosterone administered to female and male mice modulated the acoustic characteristics of USVs. There was no clear difference in acoustic characteristics between males and females. Call frequencies were higher in testosterone propionate (TP)-treated males and females compared to control males and females. When the calls were classified into nine types, there was also no distinctive difference between males and females, but TP increased the number of calls with a high frequency, and decreased the number of calls with a low frequency and short duration. The transition analysis by call type revealed that even though there was no statistically significant difference, TP-treated males and females had a similar pattern of transition to control males and females, respectively. Collectively, these results suggest that testosterone treatment can enhance the emission of USVs both in male and female, but the acoustic characteristics of TP-treated females were not the same as those of intact males.

: Testosterone masculinizes male sexual behavior through an organizational effect during the perinatal period. We previously reported that the emission of ultrasonic vocalizations (USVs) in male mice was dependent on the organizational effects of testosterone; females treated with testosterone in the perinatal period had increased USV emissions compared to males. Recently, it was revealed that male USVs have various acoustic characteristics and these variations were related to behavioral interactions with other mice. In this regard, the detailed acoustic character changes induced by testosterone have not been fully elucidated. Here, we revealed that testosterone administered to female mice during the perinatal period modulated the acoustic characteristics of USVs. There was no clear difference in acoustic characters between males and females. Call frequencies were higher in TP-treated males and females compared to control males and females. When the calls were classified into nine types, there was also no distinctive difference between males and females, but TP increased the number of calls with a high frequency, and decreased the number of calls with a low frequency and short duration. The transition analysis by call type revealed that even though there was no statistically significant difference, TP-treated males and females had a similar pattern of transition to control males and females, respectively. Collectively, these results suggest that testosterone treatment can enhance the emission of USVs in females, but the acoustic characteristics are not the same as those of intact males.