Professor The University of Tokyo Tokyo, Tokyo, Japan
Abstract Authors: Analysis of Reproductive Function in Marcksl1 Knockout Mice Fuko Matsuda1; Yukina Oshimo1; Qian Wang1; Sota Bekki1; Xiao Jia1; Takahiro Sakono1; Arisa Munetomo1; Shigeru Kakuta1; Fumie Magata1
1. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Abstract Text: Myristoylated alanine-rich C-kinase substrate like protein 1 (MARCKSL1) is a member of the MARCKS family of actin-binding proteins. However, its role in reproductive function remains unclear. In this study, we aimed to clarify its effects on fertility and gonadal function using Marcksl1 knockout mice.
Jcl:ICR mice were used to generate Marcksl1 knockout mice by the CRISPR-Cas9 system. To evaluate the fertility, wild-type (WT), heterozygous knockout (HET), and homozygous knockout (KO) animals were mated at 3–6 months of age, and births, litter size, and the proportion of each genotype in the neonates were analyzed. To evaluate the fetuses during pregnancy, HET males and HET females were mated, and litter size, the proportion of each genotype, and morphology of fetuses at embryonic day 10.5 and 18.5 were analyzed. The localization of MARCKSL1 in testes and ovaries was determined by immunohistochemistry. To examine testicular function, testes were weighed, and spermatozoa in the cauda epididymis were counted in mature males. To examine ovarian function, the number of follicles at each stage was counted in the ovaries of mature diestrus females. In addition, the number of ovulated oocytes after the induction of superovulation was counted.
Having or not having births did not vary by genotype, but the KO males × HET females and KO males × KO females combination resulted in a reduced number of neonates (6.8 ± 0.4 and 3.2 ± 1.2, respectively) compared with WT × WT combination (13.3 ± 1.4). A proportion of HET and KO fetuses had anencephaly (6–76%), a malformation that causes embryonic lethality, and the incidence of anencephaly was consistent with the rate of reduction in litter size. MARCKSL1 was strongly expressed in Sertoli cells and granulosa cells in WT males and females, respectively. Testicular weight and sperm counts did not differ among genotypes (n = 3–4). The number of primordial follicles was significantly lower in HET and KO compared with WT, while the number of primary follicles, secondary follicles, and antral follicles did not differ among genotypes (n = 4–8). The number of ovulated oocytes was significantly lower in KO compared with WT and HET (n = 13–14).
Marcksl1 deficiency did not cause infertility in either male or female mice. Marcksl1 deficiency resulted in reduced litter size, but this was thought to be due to embryonic lethality caused by the development of anencephaly in the fetus, rather than to a reduction in parental reproductive function. MARCKSL1 is expressed in Sertoli cells but is unlikely to play a significant role in testicular function. On the other hand, it has been suggested that MARCKSL1 is required for maintaining a normal number of primordial follicles and ovulation. In conclusion, MARCKSL1, although not essential for fertility, was found to be involved in ovarian function.
