Amelioration of reproduction-associated oxidative stress in a viviparous insect is critical to prevent reproductive senescence

Abstract

Impact of reproductive processes upon female health has yielded conflicting results; particularly in relation to the role of reproduction-associated stress. We used the viviparous tsetse fly to determine if lactation, birth and involution lead to damage from oxidative stress (OS) that impairs subsequent reproductive cycles. Tsetse females carry an intrauterine larva to full term at each pregnancy cycle, and lactate to nourish them with milk secretions produced by the accessory gland ( = milk gland) organ. Unlike most K-strategists, tsetse females lack an apparent period of reproductive senescence allowing the production of 8-10 progeny over their entire life span. In a lactating female, over 47% of the maternal transcriptome is associated with the generation of milk proteins. The resulting single larval offspring weighs as much as the mother at birth. In studying this process we noted an increase in specific antioxidant enzyme (AOE) transcripts and enzymatic activity at critical times during lactation, birth and involution in the milk gland/fat body organ and the uterus. Suppression of superoxide dismutase (sod) decreased fecundity in subsequent reproductive cycles in young mothers and nearly abolished fecundity in geriatric females. Loss of fecundity was in part due to the inability of the mother to produce adequate milk to support larval growth. Longevity was also impaired after sod knockdown. Generation of OS in virgin females through exogenous treatment with hydrogen peroxide at times corresponding to pregnancy intervals reduced survival, which was exacerbated by sod knockdown. AOE expression may prevent oxidative damage associated with the generation of nutrients by the milk gland, parturition and milk gland breakdown. Our results indicate that prevention of OS is essential for females to meet the growing nutritional demands of juveniles during pregnancy and to repair the damage that occurs at birth. This process is particularly important for females to remain fecund during the latter portion of their lifetime.

Figure 1. Tsetse fly investment in their progeny during lactation.

A. Changes in dry mass of single intrauterine larva throughout development. B. Predicted read abundance for the 12 major milk protein genes (milk gland protein 1–10, transferrin and acid sphingomyelinase 1) throughout lactation based on fold changes in milk proteins in relation to transcriptome analysis measured at the peak of lactation (17–18 d) and 24–48 h after parturition according to Benoit et al. . C. Total lipid content in females through pregnancy.

Figure 2. Levels of oxidative stress markers recovered from mothers through the 1^st gonotrophic cycle.

A. Lipid oxidation levels by measurement of lipid peroxidation. Samples were collected from female flies after progeny removal throughout reproduction. Mean ± SE of five groups of 3 flies. B. Protein oxidation by measurement of protein carbonyl levels. Mean ± SE of five groups of 3 flies.

Figure 3. Antioxidant gene expression and activity levels throughout tsetse pregnancy.

A. Transcript levels for Mn/Fe superoxide dismutase (Mn/Fe sod), Cu/Zn sod and catalase measured 24 h after the last blood meal. Each point represents the mean ± SE of four measurements. B. Antioxidant activity. Each sample represents the mean ± SE of four samples.

Figure 4. Effect of RNA interference of Mn/Fe sod and Cu/Zn sod on tsetse fecundity.

A. Average number of pupae produced per female during generation of the 1^st (L1), 2^nd (L2) and 3^rd (L3) pregnancy cycle after sod knockdown in the 14^th day of the fly development and 5^th day of subsequent pregnancy cycles. Mean ± SE of three groups of 15 flies. B. Length of the gonotrophic cycles analyzed under similar treatment as A. Mean ± SE of three groups of 15 flies are shown. Expression of milk gland proteins (mgp1, mgp7 and asmase1) during the early (C, 1^st instar larva present in uterus) and late stages (D, 3^rd instar larva present in uterus) of lactation after sod knockdown during the first two pregnancy cycles.

Figure 5. Oxidative stress markers recovered from females during their third pregnancy following sod knockdown during the first two reproductive cycles.

A. Protein oxidation by measurement of protein carbonyl levels. Mean ± SE of five groups of 3 flies. B. Lipid oxidation levels measured by lipid peroxidation. Samples were collected from mothers carrying a 3^rd instar larvae in their uterus. Mean ± SE of five groups of 3 flies.

Figure 6. Age-related fecundity patterns in various species in comparison to tsetse flies.

A. Medfly, (eggs/day) , , human (traditional Ache population, progeny/year) , lions (progeny/year) , Drosophila melanogaster (eggs/day) and tsetse fly (this study, [33]). B. Age-related fecundity patterns in tsetse fly after knockdown Mn/Fe sod and Cu/Zn sod. Mean ± SE for three groups of 15 flies.

Figure 7. Survival of pregnant and virgin females following sod knockdown and exogenous treatment with H₂O₂.

A. Longevity following sod knockdown in mated and unmated females. Mean ± SE of 15 flies. B. Survival of groups of virgin flies to mimic subjected to one of four treatments: H₂O or H₂O₂ injections at intervals during the peak of lactation that matched those of tsetse fly pregnancy, Mn/Fe and Cu/Zn sod during the first three pregnancy cycles and Mn/Fe and Cu/Zn sod during the first three pregnancy cycles along with H₂O₂ at intervals that match those of pregnancy. Survival data was measured using a Kaplan-Meier plot along with a log rank test. Arrows indicate treatment with H₂O or H₂O₂.

Figure 8. Population modeling following sod knockdown.

A. Frequency of growth rate. B. Reduction in growth rate between sod knockdown and control (siGFP). Results represent 10,000 simulated replicates.

Figure 9. Summary for the role of oxidative stress and antioxidant enzyme expression during tsetse fly reproduction.

Developmental images adapted from Benoit et al. . Cross sections adapted from Ma and Denlinger , Hecker and Moloo and Yang et al. .