Impact of chronic vitamin A excess on sperm morphogenesis in mice

Abstract

Background:The increasing availability of fortified foods and supplements has caused an overconsumption of vitamin A (VA), above the recommended level. To date, the effects of chronic VA excess (VAE) on spermatogenesis remain unclear.Objective: This study aims to investigate the long-term excessive intake of VA effects on spermatogenesis in mice. Materials and methods:Dams were initially fed a control diet (4 IU/g) or a VAE diet (250 IU/g), 4 weeks prior to mating and during pregnancy. Dams and their male pups continued this diet regimen until the offspring reached 12 weeks of age. At 12 weeks of age, epididymis caudal spermatozoa and testes were collected. For histological analysis, sections were stained with periodic acid-Schiff-hematoxylin, and quantitative PCR was used to detect changes in gene expression in the testes of the VAE mice. Sperm motility and morphology were evaluated to detect the endpoint of VAE toxicity.Results: Body weights were not significantly different between the control and VAE groups. Testicular cross-sections from the control and VAE mice contained a normal array of germ cells, and the daily sperm production was similar between the two groups. However, the percentage of seminiferous tubules in stages VII and VIII was significantly lower in the VAE mice than in the control. In addition, significant changes in the expression of genes involved in retinoid metabolism, spermatogenesis, and spermiogenesis were detected in the testes of the VAE mice. Consistently, sperm motility and head morphology were significantly impaired in the VAE mice. Discussion and Conclusion:Our findings suggest that long-term dietary intake of VAE was able to influence both pre-and post-meiotic spermatogenesis. As a result of testicular toxicity, we demonstrated, to the best of our knowledge, for the first time that long-term VAE caused sperm-head abnormalities.

Reactive blue 2 labels protamine in late-haploid spermatids and spermatozoa and can be used for toxicity evaluation

“…Two serial testis sections were prepared: one was used for visualization of male germ cells expressing protamine according to RB2 staining, and the other was applied for staging of the mouse seminiferous epithelium cycle using peanut agglutinin (PNA) lectin histochemistry, as previously described. 18, 19 For the former analysis, the testicular and epididymal cross-sections were stained with 0.1% RB2 in 100 mM carbonate-bicarbonate buffer (pH 10) for 30 min, and the excess dye was then washed out with the carbonate-bicarbonate buffer. For the latter, the sections were immersed in 20 mM Tris-HCl buffer (pH 9) and heated at 90°C for 30 min for antigen retrieval.…”

“…Spermatozoa were collected according to our previously reported method with minor modifications. 19, 20 Briefly, the cauda epididymis was dissected, and the connective tissue, fat pad, muscles, and vas deferens were removed. For sampling of spermatozoa, the cauda epididymis from each animal was cut using a surgical blade and then minced using small scissors in 10 mM HEPES-buffered TYH culture medium, as described above.…”

“…The copyright holder for this preprint this version posted March 8, 2023. ; https://doi.org/10.1101/2023.03.06.531276 doi: bioRxiv preprint determined to simplify the identification of the cell population, as previously reported. 18,19 Briefly, testis sections from the control mice were observed to determine the staging of the seminiferous epithelium cycle. The 12 stages (I-XII) of spermatogenesis in mice were then determined using the combination of PNA lectin histochemistry for the acrosomes of spermatids and Hoechst staining for chromatin in the nuclei, according to the established criteria of stages based on the position, size, and shape of acrosomes.…”

“…Spermatozoa were collected according to our previously reported method with minor modifications. 19,20 Briefly, the cauda epididymis was dissected, and the connective tissue, fat pad, (which was not certified by peer review) is the author/funder. All rights reserved.…”

Spatiotemporal Small Non-coding RNAs Expressed in the Germline as an Early Biomarker of Testicular Toxicity and Transgenerational Effects Caused by Prenatal Exposure to Nanosized Particles

“…Supplying retinoic acid (an active metabolite of vitamin A) to vitamin A-deficient rodents initiated both spermatogonial differentiation and meiosis in male germ cells with the induction of a germ cell-specific retinoic acid response gene, stimulated by retinoic acid gene 8 (Van Pelt and De Rooij, 1990a , b ; Anderson et al, 2008 ), as well as early spermatogenesis, followed by meiosis. We recently demonstrated that chronic vitamin A excess affects spermatogenesis and subsequently impacts sperm parameters in mice (Yokota et al, 2019 , 2021 ). This transition of undifferentiated spermatogonia into differentiated spermatogonia occurs at stages VII–VIII, and meiosis is initiated in preleptotene spermatocytes at the same stages to give rise to leptotene and zygotene spermatocytes at stages IX to XII (Oakberg, 1956a ).…”