Figure 1.
Schematic diagram summarizing timeline of experimental procedures in this study.
The presence of a vaginal plug was designated as gestational day (GD) 0. Seventy-two pregnant mice were randomly divided into two groups. In LPS group, the pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD) 13 to GD18. The normal saline-treated pregnant mice served as controls. Six pregnant mice in each group were sacrificed. Fetal outcomes were evaluated. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Testicular development, steroidogenesis and spermatogenesis were examined on PND 35 and 63, respectively.
Figure 2.
Body weights in maternal mice and its offspring.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. The dams were sacrificed on GD 18. (A) Maternal weights were recorded daily from GD13 to GD18. (B) Male and female fetuses were weighed separately on GD 18. Data were expressed as means ± SEM. *P<0.05, **P<0.01 as compared with controls.
Figure 3.
Testicular histology and serum T in male fetuses at GD 18.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. The dams were sacrificed on GD 18. Testes were collected from male fetuses at 6 h after the last LPS treatment. Testicular cross-sections from controls (A and a) and LPS-treated mice (B and b) were stained with H&E. (A and B) Magnification 50×; (a and b) Magnification 200×. Leydig cells in fetal testes from controls (C and c) and LPS-treated mice (D and d) were immunolocalized by staining with a polyclonal antibody against 3β-HSD. Arrows show 3β-HSD-positive cells. (C and D) Magnification 50×; (c and d) Magnification 200×. (E) Distribution of Leydig cell (LC) clusters in fetal testes was analyzed. Small clusters accounting for ≤5% of total LC cluster area per testis, medium clusters for 5.1–14.9% and large clusters for ≥15% of total LC cluster area per testis. (F) Serum T in male fetuses was measured by RIA. Data were expressed as means ± SEM. *P<0.05, **P<0.01 as compared with controls.
Figure 4.
Body weight and gonads weight in male mice at PND 35.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Some males each group were sacrificed on PND 35. Testes, prostates plus seminal vesicles were collected from male offsprings. (A) Body weight in male offspring was recorded from PND 4, 7, 14, 21, 28 and 35. (B) Testes and prostates plus seminal vesicles were weighed separately on PND 35. Data were expressed as means ± SEM of twelve samples from six litters. *P<0.05, **P<0.01 as compared with controls.
Figure 5.
Testicular histology and AGD in male mice during puberty.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Some males each group were sacrificed on PND 35. Testes were collected from male offsprings. Testicular cross-sections from controls (A and a) and LPS-treated mice (B and b) were stained with H&E. (A and B) Magnification 50×; (a and b) Magnification 200×. Arrowhead shows a large vacuole. Leydig cells in testes from controls (C) and LPS-treated mice (D) were immunolocalized by staining with a polyclonal antibody against 3β-HSD at magnification 100×. Arrows show 3β-HSD-positive cells. (E) The number of testicular Leydig cells was counted. Five fields were randomly selected from each section at magnification 100×. (F) Anogenital distance (AGD) in males and females was examined on PND 26. Data were expressed as means ± SEM. **P<0.01 as compared with controls.
Figure 6.
Serum T and LH in male mice at PND 35.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Some males each group were sacrificed on PND 35. Sera were collected from male offsprings. (A) Serum T in males was measured by RIA. (B) Serum LH in males was measured by ELISA. Data were expressed as means ± SEM of twelve samples from six litters. *P<0.05 as compared with controls.
Figure 7.
Gonads weight and the number of sperm in male mice at PND 63.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Some males each group were sacrificed on PND 63. Testes, prostates plus seminal vesicles were collected from male offsprings. (A) Testes and prostates plus seminal vesicles were weighed separately on PND 63. (B) The number of sperm per epididymis was counted on PND 63. Data were expressed as means ± SEM of twelve samples from six litters. *P<0.05 as compared with controls.
Figure 8.
Testicular histology in male mice at PND 63.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Some males each group were sacrificed on PND 63. Testes were collected from male offsprings. Testicular cross-sections from controls (A) and LPS-treated mice (B) were stained with H&E at magnification 50×. Arrowheads show massive sloughed germ cells in the lumen of tubules. Leydig cells in testes from controls (C) and LPS-treated mice (D) were immunolocalized by staining with a polyclonal antibody against 3β-HSD at magnification 100×. Arrows show 3β-HSD-positive cells. (E) The percent of three different stages of seminiferous tubules in total tubules was counted. Testicular cross-sections were stained by H&E. The cycle of seminiferous tubules was classified into three stage groups: stages I–VI, VII–VIII, IX–XII. Data were expressed as means ± SEM of six sections from six litters. More than 150 tubules per slide were observed. *P<0.05, **P<0.01 as compared with controls. (F) The number of testicular Leydig cells per field was counted. Five fields were randomly selected from each section at magnification 100×. Data were expressed as means ± SEM of six sections from six litters.
Figure 9.
Serum T and LH in male mice at PND 63.
Maternal mice were injected with LPS (50 µg/kg, i.p.) daily from gestational day (GD) 13 to GD18. Within 24 h after birth, excess pups were removed, so that four males and four females were kept per dam. At postnatal day (PND) 21, pups were separated from the siblings and housed five to a cage. Some males each group were sacrificed on PND 63. Sera were collected from male offsprings. (A) Serum T in males was measured by RIA. (B) Serum LH in males was measured by ELISA. Data were expressed as means ± SEM of twelve samples from six litters. *P<0.05 as compared with controls.