Rats undernourished in utero have altered Ca2+ signaling and reduced fertility in adulthood

Abstract

Epidemiological and animal studies have shown that placental undernutrition impairs reproduction in adult offspring, but the underlying molecular mechanisms within the male genital tract remain unknown. Due to its special physiological characteristics in transport and the modulation of the environment to which its luminal content is exposed, we hypothesized that the vas deferens would be a highly sensitive target. The goals were to investigate whether intrauterine malnutrition affects molecular mechanisms related to Ca(2+)- and oxidative stress-modulated processes and causes structural alterations in the adult rat vas deferens that could attenuate fecundity and fertility. Male adult rats malnourished in utero had increased vas deferens weight associated with thickening of the muscular coat, a decrease in the total and haploid germ cells, a marked increase in the immature cells, and a decline in the numbers of pregnant females and total offspring per male rat. The ex vivo response of vas deferens from malnourished rats demonstrated an accentuated decrease in the contractile response to phenylephrine. The vas deferens had a marked decrease in Ca(2+) transport due to the uncoupling of Ca(2+)-stimulated ATP hydrolysis and ATP-driven Ca(2+) flux, and the downregulation of both sarco-endoplasmic reticulum Ca(2+)-ATPase 2 and the coupling factor 12-kDa FK506-binding protein. An increase in protein carbonylation (a marker of oxidative damage) and an imbalance between protein kinases C and A were observed as a legacy of undernutrition in early life. These results provide the structural and molecular basis to explain at least in part how maternal undernutrition affects fecundity and fertility in adult male rats.

Keywords: Intrauterine malnutrition; oxidative damage markers; reproductive performance; vas deferens calcium handling.

Figure 1

Intrauterine malnutrition reduces Ca²⁺ accumulation by vesicles derived from vas deferens membranes. Steady-state ATP-dependent Ca²⁺ accumulation in the lumen of vesicles derived from membranes of whole vas deferens homogenates was measured in control (CTRL) and intrauterine malnourished rats (IM) after 120 min. The results are the mean ± SEM (n = 4 homogenates obtained with 2–3 pairs of vas deferens of rats from different litters); *P = 0.0018 compared with the CTRL group (assessed by unpaired Student’s t-test).

Figure 2

Fetal malnutrition increases the response to Ca²⁺ for PMCA and SERCA activities from the vas deferens. (A) Initial rate of total P_i release: Ca²⁺-ATPase activity was measured at 5 min in homogenates from CTRL (empty bars) and IM rats (black bars). The results are the mean ± SEM (n = 3–5 homogenates obtained as described in the legend to Fig.1); *P < 0.0001 with respect to CTRL. (B) Total P_i release at the time of Ca²⁺ uptake measurements (120 min). Groups and symbols as in (A). The results are the mean ± SEM (n = 3–5 homogenates obtained as described in the legend to Fig.1); *P = 0.0002 with respect to CTRL. Differences in (A) and (B) were assessed by unpaired Student’s t-test. (C and D) Activity of PMCA and SERCA were measured at 120 min in the presence of the Ca²⁺ concentrations shown. CTRL: empty bars; IM: black bars. The results are the means ± SEM (n = 3–5 homogenates as above). In (C and D): *P < 0.0001 in all cases compared to the corresponding CTRL at 1, 100, and 10,000 nmol/L Ca²⁺, respectively. Differences were assessed by one-way ANOVA followed by the Bonferroni post-test for selected pairs. PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarco-endoplasmic reticulum Ca²⁺-ATPase; CTRL, control; IM, intrauterine malnourished; ANOVA, analysis of variance.

Figure 3

Undernutrition-induced modifications in the abundance of Ca²⁺-handling proteins (PMCA, SERCA2, and FKBP12) in the vas deferens. (A) PMCA. Upper panel: representative immunoblotting. Lower panel: graphic representation of four immunoanalyses performed using different preparations (homogenates obtained with 2–3 pairs of vas deferens of rats from different litters) and a primary antibody from Santa Cruz Biotechnology (catalog number sc-28765). Samples from CTRL and IM were analyzed in parallel in the same gel (10% acrylamide). (B) SERCA2. Upper panel: representative immunoblotting. Lower panel: graphic representation of four immunoanalyses (homogenates obtained with 2–3 pairs of vas deferens from rats from different litters) and a primary antibody from Sigma-Aldrich (catalog number s1314). *P = 0.0005 versus the corresponding CTRL, as assessed by unpaired Student’s t-test. (C) FKBP12. Upper panel: representative image of three immunoblottings, which were performed using different preparations as above and a primary antibody from Santa Cruz Biotechnology (catalog number sc-28814). Lower panel: graphic representation of the three immunoanalyses. The data are expressed as the percentage of CTRL (mean ± SEM). *P = 0.009 versus the corresponding CTRL (unpaired Student’s t-test). PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarco-endoplasmic reticulum Ca²⁺-ATPase; FKBP12, 12-kDa FK506-binding protein; CTRL, control; IM, intrauterine malnourished; NS, not significant.

Figure 4

In utero malnutrition increases carbonylation but not lipid peroxidation or free sulfhydryl (-SH) content. (A) Levels of lipid peroxidation (n = 3 pairs of vas deferens; all rats coming from different litters). (B) Protein carbonylation (n = 6 homogenates obtained with 2–3 pairs of vas deferens of rats from different litters). (C) Total free -SH group content (n = 3 homogenates as above). The results are expressed as the mean ± SEM. In B, *P = 0.039 versus CTRL (unpaired Student’s t-test). CTRL, control; NS, not significant.

Figure 5

Intrauterine malnutrition upregulates PKA and PKCλ and decreases the PKA/PKC activities ratio. (A) PKA α-subunit content. Upper panel: representative immunoblotting; lower panel: graphic representation of three immunoanalyses (10% acrylamide) performed using homogenates obtained with 2–3 pairs of vas deferens from rats from different litters and a primary antibody from Santa Cruz Biotechnology (catalog number sc-903). The data are the percentages of the CTRL. *P = 0.012 versus the corresponding CTRL. (B) PKA activity. The results are the mean ± SEM (n = 4 homogenates obtained with 2–3 pairs of vas deferens from rats of different litters in both groups). *P = 0.013 versus the corresponding CTRL. (C–F) PKCα, PKCε, PKCζ, and PKCλ contents. In all panels, representative immunoblotting is in the upper section; the graphic representation of four immunoanalyses performed using different homogenates obtained as above is in the bottom section. The primary antibodies were from Santa Cruz Biotechnology (catalog numbers sc-208, sc-214, sc-216, and sc-1091, respectively). *P = 0.005 versus the corresponding CTRL. (G) Calphostin-sensitive PKC activity. The results are the mean ± SEM (n = 4 different homogenates obtained as above in both groups). *P = 0.018 versus the respective CTRL. In (A–G), the differences were assessed by unpaired Student’s t-test. (H) PKA/PKC activities ratio. PKA, AMP-dependent protein kinase; PKC, 4 protein kinase C; CTRL, control.

Figure 6

Representative photomicrographs (Masson’s trichrome) of the vas deferens. Epididymal (A and B) and prostatic (C and D) segments from CTRL (A and C) and IM rats (B and D). Key: l, lumen; m, mucosa; mc, muscular coat (keys are indicated only in A for simplicity). Representative images obtained from six different rats (all coming from different litters) from each group. The numbers below the panels (mean ± SEM in μm²) are the corresponding values for MCA obtained from histomorphometric analyses of the six vas deferens mentioned above. *P = 0.041 when the epididymal portion from IM rats (B) is compared with the corresponding CTRL (A); *P = 0.0004 when the prostatic portion from IM rats (D) is compared with the corresponding CTRL (C) (unpaired Student’s t-test). CTRL, control; IM, intrauterine malnourished; MCA, muscular coat area.

Figure 7

Intrauterine malnutrition affects total, haploid, and immature cell counts in the testis, epididymis, and vas deferens. Total (A) and haploid (B) cells were counted in the testis, epididymis, and vas deferens from control (empty bars, n = 10 rats, all from different litters) and intrauterine malnourished rats (black bars, n = 7 rats, all from different litters). (C) Percentage of immature cells in the total cell populations. The results are the mean ± SEM. *P values are indicated above the bars (unpaired Student’s t-test).

Figure 8

Contractile response of isolated epididymal portions of vas deferens at increasing concentrations of phenylephrine. The percent increase in contraction from baseline was recorded based on the concentrations of the α1-adrenoceptor agonist phenylephrine shown on the abscissa. Equation 1 (see Methods section) was adjusted to the experimental points from CTRL (○) or IM rats (●). The figure depicts the fitting of the function of the mean values. The EC₅₀ and Hill slope values were calculated by averaging values obtained in each single fitting. Three to four CTRL and IM male offspring randomly selected from the litters of two CTRL and two IM dams were used, thus giving n = 2 for each group. Overall, the mean values of the determinations in quadruplicate (one CTRL and one IM mother) and triplicate (one CTRL and one IM mother) agreed within 6% (CTRL pair) and 12% (IM pair). CTRL, control; IM, intrauterine malnourished.

Figure 9

In utero malnutrition reduces the reproductive performance of adult male rats. Fertility, left axis: number of well-nourished pregnant females resulting from mating with one CTRL or one IM male rat. Fecundity, right axis: offspring from each CTRL or IM male rat. The data are the mean ± SEM of matings from six groups of one male (CTRL or IM) with three females each (fertility) or the mean ± SEM of offspring from six mated groups with one CTRL or one IM male rat (fecundity); n = 6, corresponding to males (CTRL or IM) coming from different litters, originally with eight pups each. *P = 0.002 (fertility) and *P = 0.005 (fecundity) versus the corresponding CTRL (unpaired Student’s t-test). CTRL, control; IM, intrauterine malnourished.