Lin28a regulates germ cell pool size and fertility

Abstract

Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males, the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild-type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage.

Fig. 1

Lin28a expression in normal gonadal tissues at different ages. (A) E13.5 genital ridge. (B) Newborn ovary. (C) 5-week-old ovary. (D) Newborn testis. (E) 8-month-old testis. Insets show higher magnification of Lin28a positive cells. Note that Lin28a expression in cytoplasm. Scale bars 100 µm.

Fig. 2

Lin28a deficiency reduces germ cell pool size. (A) Percentage of primordial germ cells in Lin28a −/− and control genital ridges at E13.5. N=4–15. (B) let-7 levels of primordial germ cells in E13.5 embryos. N=3. (C) Lin28a +/+ and −/− newborn ovaries. (D) Number of Mvh+ germ cells in Lin28a +/+ and −/− newborn ovaries. N=4–5. (E) Lin28a +/+ and −/− newborn testes. (F–G) Number of Mvh+ germ cells per tubule (F) and % Ki67+ germ cells (G) in Lin28a +/+ and −/− newborn testes. N=64–74 from 3–5 mice in (F) and N=3–4 in (G). * p<0.05, ** p<0.01. Error bars represent SEM. Scale bars 100 µm.

Fig. 3

Lin28a knockout females have reduced fertility. (A) A representative photographs of 5-month-old Lin28a +/− and −/− mice and their ovaries. Bar 5mm. (B–C) Ovary weight (B), ovary weight relative to body weight (C) in 5-month-old Lin28a +/− and −/− females. N=4–6. (D) H&E staining (top) and immunostaining with Mvh (bottom) of 3-month-old adult ovaries. Inset: higher magnification of primordial follicles. Note that corpus luteum (CL) are found in both Lin28a +/− and −/− ovaries. Scale bars 100 µm. (E) Number of different stages of ovarian follicles per 1 mm² in 3-month-old Lin28a +/+, +/− and −/− ovaries. N=8–10. (F) Number of live oocyte collected from 2-month-old Lin28a +/− and −/− females after superovulation. N=5–6. (G–I) Female fertility tests at 2–3 months old. Number of deliveries (G) and number of pups born (H) per Lin28a +/− and −/− female during 10-week experimental period. (I) Litter size of Lin28a +/− and −/− females. N=3–4. * p<0.05, ** p<0.01. Error bars represent SEM.

Fig. 4

Partial recovery of germ cell pool size in adult Lin28a KO males. (A) A representative photographs of 5-month-old Lin28a +/− and −/− mice and their testes. Bar 5mm. (B–F) Testis weight (B), testis weight relative to body weight (C) in 5- to 6-month-old Lin28a +/− and −/− mice. N=6. (D–F) Total epididymal sperm count per mouse (D), percentage of motile sperms (E), and percentage of abnormal sperms (F) in 7- to 8-month-old Lin28a +/− and −/− mice. N=3–4. (G) H&E and immunostaining of 6-month-old Lin28a +/− and −/− testes. (H–I) Number of Mvh+ germ cells per tubule (H) and number of tubules containing Ki67+ cells (I) in 6- to 8-month-old Lin28a +/− and −/− adult testes. N=5. ** p<0.01. Error bars represent SEM. Scale bars 100 µm.

Fig. 5

Lin28a −/− males are subfertile. (A–F) Male fertility tests at 7–10 weeks old (A–C) and 7–8 months old (D–F). Number of deliveries (A and D) and number of pups born (B and E) per Lin28a +/+, +/− and −/− male during 13-week experimental period. (C and F) Litter size of Lin28a +/+, +/− and −/− males. N=2–3. (G) Number of plugs per 5-month-old Lin28a +/+, +/− and −/− male during 4-week experimental period. N=3–6. (I–K) Plasma testosterone (I), LH (J), and FSH (K) levels in Lin28a +/+, +/− and −/− males. N=4–10. * p<0.05, ** p<0.01. Error bars represent SEM.

Fig. 6

Embryonic overexpression of let-7 miRNA reduces germ cell pool size. (A) let-7 levels of primordial germ cells in E13.5 embryos. N=3. (B) iLet-7 and control newborn testes and ovaries. (C) Number of Mvh+ germ cells per tubule in iLet-7 and control newborn testes. N=286–303 from 3 mice. (D) Number of Mvh+ germ cells per 1000 µm² in iLet-7 and control ovaries. N=3–5. * p<0.05, ** p<0.01. Error bars represent SEM. Scale bars 100 µm.