Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling

Abstract

Approximately half of all infertility cases can be attributed to male reproductive dysfunction for which low sperm count is a major contributing factor. The current study identified receptor-mediated lysophosphatidic acid (LPA) signaling as a new molecular component influencing male fertility. LPA is a small signaling phospholipid, the effects of which are mediated through at least five G protein-coupled receptors, named LPA 1-5. LPA1/2/3, but not LPA4/5, show high expression in mouse testis. Mice deficient in LPA1/2/3 showed a testosterone-independent reduction of mating activity and sperm production, with an increased prevalence of azoospermia in aging animals. A significant increase of germ cell apoptosis also was observed in testes. Germ cell apoptosis led to a reduction in germ cell proliferation. These data demonstrate a novel in vivo function for LPA signaling as a germ cell survival factor during spermatogenesis.

FIG. 1.

Messenger RNA expression of LPA receptors in WT testis. a) Quantification by real-time RT-PCR of Lpar1 through Lpar5 in 4-wk-old testes (n = 3). Error bars represent the standard deviation. Actb was used as a loading control. b) In situ localization of Lpar1, Lpar2, and Lpar3 in 15-day-, 4-wk-, and 4-mo-old testes. Lpar3 sense probes were used as a negative control. AS, Antisense; S, sense. Bar = 100 μm.

FIG. 2.

Sperm count of Lpar1, Lpar2, and Lpar3 single- and double-knockout (DKO) mice, occurrence of hematomas, and litter size of Lpar1/2/3 TKO mice. a) Comparison of sperm count in the cauda epididymis of WT and Lpar1, Lpar2, and Lpar3 single-null testes as well as Lpar1/2, Lpar1/3, and Lpar2/3 double-null testes at 6-mo of age. Sperm count is expressed as sperm number per milligram of cauda epididymis. Error bars represent the SEM (n = 5–10). Two-tail unequal variance t-test: *P < 0.005, **P < 0.001 vs. WT; ^#P < 0.05 vs. single nulls. b) Receptor-dependent occurrence of hematomas in embryos (E; Embryonic Days 11.5–18.5) and neonatal pups (N). c) Litter sizes from mating studies. The x-axis represents females (F) × Males (M). Error bars represent the SD (n = 12–26). Two-tail unequal variance t-test: *P = 1.00 × 10⁻⁶. d) Litter size at Postnatal Day (P) 0 (total and alive) and P15 (alive) from WT × WT (n = 15) and TKO × TKO (n = 114) mating. Error bars represent the SD. Two-tail unequal variance t-test: *P = 8.00 × 10⁻¹², 5.64 × 10⁻¹³, and 5.03 × 10⁻¹³, respectively, vs. WT; two-tail paired t-test: ^#P = 2.61 × 10⁻¹⁶ vs. Lpar1/2/3 TKO P0 total; ^##P = 4.83 × 10⁻¹¹ vs. Lpar1/2/3 TKO P0 alive.

FIG. 3.

Mating activity of Lpar1/2/3 TKO mice. a) Plugging rate. WT: 57/57 = 100%; TKO: 30/45 = 66.7%. Chi-square test: *P < 0.00005. b) Plug latency. WT: n = 30; TKO, n = 12. Error bars represent the SEM. Two-tail unequal variance t-test: *P ≤ 0.05. c) Pregnancy rate from plugged females. WT: 25/57 = 43.3%, TKO: 12/30 = 40%.

FIG. 4.

Histology of Lpar1/2/3 TKO testes during development: (a) Postnatal Day (P) 0, (b) P15, (c) 4 wk; (d) 6 mo. Paraffin sections (thickness, 5 μm) were cut and stained with hematoxylin and eosin. Bar = 100 μm.

FIG. 5.

Testis weight and sperm count of Lpar1/2/3 TKO males. a) Testis weight. Two-tail unequal variance t-test: *P < 0.05. b) Comparison of sperm counts in the cauda epididymis of WT and Lpar1/2/3 TKO mice at 2, 4, 6, and 8 mo of age. Sperm count is expressed as sperm number × 10⁶ per milligram of cauda epididymis. Error bars represent the SEM (n = 5–16). Two-tail unequal variance t-test: *P < 0.0005. c) The percentage of Lpar1/2/3 TKO testes with azoospermia (n = 10–20).

FIG. 6.

MAPK3/1 phosphorylation in Lpar1/2/3 TKO testicular primary cells. a) Dose response and time course of LPA-induced MAPK3/1 phosphorylation in control testis primary cells. b) Loss of LPA-induced MAPK3/1 phosphorylation in Lpar1/2/3 TKO testicular primary cells measured after LPA and sphingosine 1-phosphate (S1P) treatment for 10 min.

FIG. 7.

Germ cell apoptosis in Lpar1/2/3 TKO testes. a) ISEL⁺ detection of apoptotic cells in WT and Lpar1/2/3 TKO testes at Postnatal Day (P) 15 and 3 mo (3M). Red arrows indicate apoptotic germ cells. Green brackets indicate apoptotic spermatogonia. b) Percentages of apoptotic germ cells in control and Lpar1/2/3 TKO testes at P15, 3M, and 8 mo (8M). Error bars represent the SD (n 4–8). Two-tail unequal variance t-test: *P < 0.05. Bar = 100 μm.

FIG. 8.

Germ cell proliferation in Lpar1/2/3 TKO testes. a) BrdU detection of proliferating germ cells in WT and Lpar1/2/3 TKO testes at Postnatal Day (P) 15 and 3 mo (3M). b) Semiquantification of germ cell proliferation at P15, 3M, and 8 mo (8M). Error bars represent the SD (n = 4–8). At P15, no significant differences were found. Two-tail unequal variance t-test: *P < 0.05. Bar = 100 μm.