A lack of contact of sperm with accessory sex gland secretions deregulates DNA methylation and imprinted gene expression in rodent embryos.

Increased oxidative DNA damage is observed in sperm devoid of contact with accessory sex gland (ASG) secretion. After fertilization, these sperm may produce abnormal embryos. In this study, we investigated the possibility that the pattern of DNA methylation and imprinted gene expression in these embryos may be perturbed. Epididymal sperm, uterine sperm, and embryonic day 13 (E13) embryos were collected from hamster and mouse. The extent of global DNA methylation was determined with an antibody against methylcytosine using an embryo DNA dot. The sperm and embryo Gtl2 promoter and H19 differential methylated region (DMR) were subject to bisulfite sequencing. Expression of their reciprocally activated genes Dlk1 and Igf2 was quantified by real-time PCR. Genome-wide DNA hypo-methylation in both hamster and mouse embryos sired by males without ASG was observed. The imprinting pattern of fetal mouse Gtl2 promoter and fetal hamster H19 DMR were also disrupted while the expression of Dlk1 and Igf2 was dysregulated in the hamster embryo. This study suggests that a lack of contact of sperm with the ASG secretion disrupts genome-wide DNA methylation and also affects the DNA methylation pattern of imprinted genes in embryos.

Absence of paternal accessory sex gland secretions disturbs epigenetic reprogramming and expression of Igf2 and Dlk1 in golden hamster embryos.

Accessory sex gland (ASG) secretion is known to exert an effect on sperm that is heritable in hamster embryos. We hypothesized that ASG secretion changes the sperm epigenome, which in turn is propagated in sired embryos. To test our hypothesis, we produced male hamsters that were devoid of either all ASG (TX) or only the ventral lobe of the prostate gland (VPX). A sham-operated control group (SH) was also established. These males were mated with normal females; uterine sperm, fertilized oocytes, and pre-implantation embryos were harvested from the females after mating. Epididymal sperm were collected at the end of experiments. Immunofluorescent staining was performed on these harvested specimens using antibodies against 5-methylcytosine, Dnmt1, Dnmt3a, Dnmt3b, protamine 1, protamine 2, and aectyl-H4K5. Expression of Igf2 and Dlk1 were analyzed by real-time RT PCR and in situ hybridization. We demonstrated that the DNA methylation pattern changed dynamically in SH, TX, and VPX fertilized oocytes. In VPX and TX embryos, DNA demethylation was slower and remethylation was delayed when compared with SH embryos. In addition, Dnmt3b expression was also abnormal. When sperm from VPX and TX males were exposed to whole ASG secretion in vivo, the resulting embryos all methylated normally. Immunofluorescent staining revealed that there was no difference in protamine packaging of uterine sperm from VPX and TX males. The staining also showed a lower level of acetyl-H4K5 expression in the male pronuclei of TX produced embryos. Furthermore, the VPX and TX embryos also expressed higher levels Igf2, and Dlk1. We concluded that interactions between ASG and sperm affected: (1) histone acetylation in male pronuclei; (2) DNA methylation in fertilized oocytes; and (3) Igf2 and Dlk1 expression embryos.

Tissue specific expression and sequence analysis of a stress responsive gene Bre in adult golden hamster (Mesocricetus auratus).

Bre (brain and reproductive organ-expressed) is a new and putative stress-modulating gene of yet unknown function. BRE has previously been shown to interact with type 1 tumor necrosis factor receptor (TNFR1) and modulate the action of TNF. Apart from the brain and reproductive organs, Bre and BRE are highly expressed in steroid producing tissues such as the adrenal gland. Here we report for the first time the cloning of the Bre gene from golden hamster, a model organism extremely valuable for reproduction and steroid research, and examination of its tissue specific expression. Sequence analysis demonstrated that the peptide sequence of BRE in hamster shares approximately 99% homology with those of human, monkey and mouse. The hamster Bre gene transcribed an approximately 1.8-kb mRNA which translated a 44-kDa protein. Bre was strongly expressed in neurons and luminal epithelia of urogenital, digestive and respiratory organs. Bre was also detected in lymphoid tissues and endocrine glands. Immunohistochemistry demonstrated a similar protein expression pattern. Exceptions to this included the adrenal gland, where a high level of Bre was accompanied by weak immunoreactivity; as well as the oocytes and islets of Langerhans, where BRE protein but not the mRNA was localized. These data indicated that Bre gene products were expressed in a wide variety of tissues other than the brain and reproductive organs, as was originally described. Based on our findings, we propose that Bre is a housekeeping gene in tissues that are constantly subjected to environmental hazards such as luminal epithelia. Our results further support the proposed role for BRE in endocrine and immune functions.

Embryos sired by males without accessory sex glands induce failure of uterine support: a study of VEGF, MMP and TGF expression in the golden hamster.

To account for reproductive failure induced by surgical deletion of paternal accessory sex glands in the golden hamster in vivo, we studied expression of vegf, FLT-1 (VEGF-R1), FLK-1 (VEGF-R2), MMP and TGF-beta in endometrium of the dam and sired embryos during 5-7 days post coitum by immunohistochemistry, in situ hybridisation, semiquantitative RT-PCR and enzyme-linked immunosorbent assay. Spatiotemporal pattern of vegf expression in the control animals was similar to that reported for intact animals by our group. Removal of paternal ampullary glands did not disturb the normal expression pattern. Removal of ventral prostate glands alone or all accessory sex glands was associated with reduction of vegf transcripts and protein levels in both the embryo and endometrium. FLT-1, FLK-1 and MMP-2 were also reduced. MMP-1 was not changed whereas TGF-beta1 expression was enhanced. There was no expression in endometrium in between implantation sites. Thus the implanted embryos had a trophic effect on growth factor production by the endometrium, and the levels of expression were determined by viability and structural integrity of the conceptus. Based on these findings we concluded that incompetent embryos sired by males without the ventral prostate gland or all accessory sex glands reduced the potential of the uterus to support pregnancy. A negative cycle of events was thus set up and eventually led to premature termination of pregnancies.

c-Fos protein in photoreceptor cell death after photic injury in rats.

PURPOSE: To examine the involvement of c-Fos protein in light-induced photoreceptor cell death in rats. METHODS: Thirty-two Lewis albino rats were exposed to green fluorescent light (480-520 nm) of 300 to 320 foot-candles (3228-3443.2 lux) for 3 hours, allowed to recover in the dark, and euthanatized at 0, 1, 3, 6, 12, 24, or 96 hours after light exposure. c-Fos was detected immunohistochemically and nicked DNA by in situ TdT-dUTP terminal nick-end labeling (TUNEL). Double labeling of c-Fos and DNA nicks was also performed. RESULTS: There was a time-dependent change in the number of c-Fos-positive photoreceptor nuclei after light injury, which paralleled the change in the number of TUNEL-positive nuclei. The temporal and spatial appearance of these nuclei also matched the appearance of pyknotic nuclei of the outer nuclear layer. Double-labeling study revealed that some c-Fos-positive nuclei were also TUNEL-positive nuclei. CONCLUSIONS: There was an acute accumulation of c-Fos protein in photoreceptors associated with cell death. This study further supports other studies showing that c-Fos is linked to apoptotic photoreceptor cell death.

Glycoconjugates of the rat ciliary body epithelium: a lectin histochemical and protein blotting study.

The present study sought to identify and partially characterize the glycoconjugates specific to the double-layered ciliary body epithelium of the rat eye by lectin histochemistry and lectin blottings. Hydrated paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with a panel of 21 different biotinylated lectins, followed by streptavidin-peroxidase and the glucose oxidase-diaminobenzidine-nickel staining procedure. The results of lectin histochemistry revealed that the inner epithelial layer was rich in GlcNAc(beta1,4)GlcNAc, alpha-Gal, Gal(beta1,3)GalNAc, GalNAc(alpha1,3)GalNAc/Gal, GalNAc(alpha1,6)Gal, Fuc(alpha1,2)Gal(beta1,4)GlcNAc and Gal(beta1,4)GlcNAc(beta1,2)Man(alpha1,6) sugar residues as shown by its positive reactivities with S-WGA, PWA, DSA, GS-I-B4, PNA, DBA, SBA, WFA, UEA-I, LTA and PHA-E. The reactivities of GS-I-B4, PNA, DBA and SBA were restricted to the inner layer at the tips of the ciliary processes. On the other hand, the outer epithelial layer was stained evenly by DSA and Jacalin, and partly by MAA, showing that this epithelial layer was rich in GlcNAc(beta1,4)GlcNAc, Gal(beta1,3)GalNAc and NeuAc(alpha2,3)Gal disaccharides. These lectin binding patterns of the ciliary body epithelium suggest a topographical and functional difference in this double cell-layered epithelium. Their possible roles in the secretion of aqueous humour and production of ciliary zonule are discussed. Some identified lectin markers specific to these two cell layers may be useful for further experimental studies. Glycoproteins extracted from the dissected ciliary body were separated by SDS-PAGE electrophoresis and analyzed by protein blottings with 8 different lectins. The results showed that at least 10 major membrane-bound glycoproteins, with molecular weights ranging from 30 to 150 kD, rich in beta-GlcNAc, beta-Gal, alpha/beta-GalNAc and NeuAc(alpha2,6)Gal residues, were present in the microsomal fraction.

Transient patterns of acetylcholinesterase activity in developing thalamus: a comparative study in rodents.

This paper describes acetylcholinesterase (AChE) activity in the dorsal thalamus of several rodents, including rat, mouse, gerbil, hamster and guinea pig. Tissue from fetal, neonatal, and adult animals was studied using histochemical techniques. Developing animals of all species display prominent AChE staining in the ventral medial geniculate nucleus and the ventral posterior nucleus, while adult animals show very light staining in these thalamic regions. Similarly, the dorsal lateral geniculate nucleus of all species shows stronger staining in developing animals than in adults. These data indicate that transient expression of AChE activity in primary sensory thalamic nuclei is a characteristic common to a variety of rodents.

Neonatal enucleations reduce number, size, and acetylcholinesterase histochemical staining of neurons in the dorsal lateral geniculate nucleus of developing rats.

Previous studies have demonstrated that transient patterns of acetylcholinesterase (AChE) activity are characteristic of geniculo-recipient regions of rat cortical area 17 during the second and third postnatal weeks of life. Neonatal enucleation results in a marked reduction of this transiently expressed cortical AChE. Parallel studies have demonstrated that the dorsal lateral geniculate nucleus (dLGN) also expresses AChE transiently during development. The present study examines neuronal number and size as well as AChE histochemical staining in the dLGN of normal and neonatally enucleated rat pups to determine whether changes in dLGN neurons could account for the decreased visual cortical AChE staining that results from neonatal enucleation. Changes in 4 parameters in dLGN were noted after neonatal enucleation. First, a 26-37% shrinkage in the volume of dLGN occurred contralateral to enucleation. Second, enucleation resulted in a loss of 16-30% of AChE-stained neuronal somata. Third, remaining AChE-positive neuronal somata appeared shrunken by approximately 40%. Fourth, intensity of AChE histochemical staining of individual dLGN neurons was reduced by approximately 24% following neonatal enucleation. These data suggest that loss of transient AChE activity in cortical area 17 consequent to neonatal enucleation is secondary to enucleation-induced alterations in the dLGN; these alterations include loss of neurons, shrinkage of neurons, and an apparent decrease in the ability of neurons to synthesize AChE. These data support the hypothesis that geniculocortical projection neurons express AChE transiently during development of geniculocortical connectivity and indicate that normal afferent connections and/or activity are important for the transient expression of AChE by these neurons.