Small-interfering RNAs have been used to disrupt the function of the more than 100 copies of the Sly gene on the mouse Y chromosome, leading to defective sex chromosome repression during spermatid differentiation and, as a consequence, sperm malformations and near-sterility.
To examine effects of nutrient restriction and dietary Se on maternal and fetal visceral tissues, 36 pregnant Targhee-cross ewe lambs were allotted randomly to 1 of 4 treatments in a 2 x 2 factorial arrangement. Treatments were plane of nutrition [control, 100% of requirements vs. restricted, 60% of controls] and dietary Se [adequate Se, ASe (6 microg/kg of BW) vs. high Se, HSe (80 microg/kg of BW)] from Se-enriched yeast. Selenium treatments were initiated 21 d before breeding and dietary restriction began on d 64 of gestation. Diets contained 16% CP and 2.12 Mcal/kg of ME (DM basis) and differing amounts were fed to control and restricted groups. On d 135 +/- 5 (mean +/- range) of gestation, ewes were slaughtered and visceral tissues were harvested. There was a nutrition x Se interaction (P = 0.02) for maternal jejunal RNA:DNA; no other interactions were detected for maternal measurements. Maternal BW, stomach complex, small intestine, large intestine, liver, and kidney mass were less (P < or = 0.01) in restricted than control ewes. Lung mass (g/kg of empty BW) was greater (P = 0.09) in restricted than control ewes and for HSe compared with ASe ewes. Maternal jejunal protein content and protein:DNA were less (P < or = 0.002) in restricted than control ewes. Maternal jejunal DNA and RNA concentrations and total proliferating jejunal cells were not affected (P > or = 0.11) by treatment. Total jejunal and mucosal vascularity (mL) were less (P < or = 0.01) in restricted than control ewes. Fetuses from restricted ewes had less BW (P = 0.06), empty carcass weight (P = 0.06), crown-rump length (P = 0.03), liver (P = 0.01), pancreas (P = 0.07), perirenal fat (P = 0.02), small intestine (P = 0.007), and spleen weights (P = 0.03) compared with controls. Fetuses from HSe ewes had heavier (P < or = 0.09) BW, and empty carcass, heart, lung, spleen, total viscera, and large intestine weights compared with ASe ewes. Nutrient restriction resulted in less protein content (mg, P = 0.01) and protein:DNA (P = 0.06) in fetal jejunum. Fetal muscle DNA (nutrition by Se interaction, P = 0.04) concentration was greater (P < 0.05) in restricted ewes fed HSe compared with other treatments. Fetal muscle RNA concentration (P = 0.01) and heart RNA content (P = 0.04) were greater in HSe vs. ASe ewes. These data indicate that maternal dietary Se may alter fetal responses, as noted by greater fetal heart, lung, spleen, and BW.
Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.
The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.
Slx and Slxl1 are genes present in multiple copies on the mouse X chromosome. Using transgenically-delivered small interfering RNAs to disrupt their function, we show that Slx and Slxl1 are important for normal sperm differentiation and male fertility.
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