Ad libitum feeding alters mRNA abundance in the ovarian cortex of broiler breeder hens.

Ad libitum: feeding in broiler breeder (BB) hens causes reduced egg production, lower fertility, and improper eggshell deposition. Restricted feeding (RF) is the only effective intervention available to normalize ovarian function and improve reproductive efficiency. This study aimed to assess the transcriptional changes in ovarian cortex of BB hens with free access to feed compared to those on a RF diet. RNA was isolated from the ovarian cortex of Cobb 500 pullets raised to 10 and 16 weeks of age on either a full-feeding (FF) or RF diet. Microarray analysis identified 386 differentially expressed genes between the two feeding groups at 16 weeks of age. Gene ontology enrichment identified overrepresentation of Neuroactive ligand-receptor interaction pathways, Cell adhesion molecules, Steroid hormone biosynthesis, and various KEGG pathways. From these groups, 46 genes were selected for follow-up validation by quantitative PCR. The findings show that 33 of the 46 genes had significantly different abundance by age and/or feeding level. Most of these genes were repressed in RF hens and belonged to the steroid biosynthesis and neuropeptide signaling groups. The VIPR2 receptor was higher in the FF group leading us to hypothesize that vasoactive intestinal peptide (VIP) is an important regulator of small cortical follicles. Culture of hen cortical follicles with VIP increased Star, an indication of increased steroidogenic activity, although did not elevate Cyp11a1. These results offer insights and suggest the possible mechanisms and pathways responsible for the increases in cortical follicle growth associated with excess feed intake in BB hens. Lay summary: Giving breeder hens unrestricted access to feed can lead to problems with their ovaries, including excessive growth of the ovary and reduced fertility. Giving a limited amount of feed is the only effective way to reduce this growth of the ovaries and improve fertility. This study aimed to assess the changes in the molecules that make proteins in the body in hens fed unrestricted and restricted diets. In the hens fed a limited amount of feed, there were more of one type of molecules, while there were more of another type in the ovaries of hens with unrestricted access to feed. These results show that how much a hen eats can alter the number of these molecules in the ovary and this could help us understand why their ovaries grow excessively and why their eggs are less fertile.

Advanced age increases frequencies of de novo mitochondrial mutations in macaque oocytes and somatic tissues.

Mutations in mitochondrial DNA (mtDNA) contribute to multiple diseases. However, how new mtDNA mutations arise and accumulate with age remains understudied because of the high error rates of current sequencing technologies. Duplex sequencing reduces error rates by several orders of magnitude via independently tagging and analyzing each of the two template DNA strands. Here, using duplex sequencing, we obtained high-quality mtDNA sequences for somatic tissues (liver and skeletal muscle) and single oocytes of 30 unrelated rhesus macaques, from 1 to 23 y of age. Sequencing single oocytes minimized effects of natural selection on germline mutations. In total, we identified 17,637 tissue-specific de novo mutations. Their frequency increased ∼3.5-fold in liver and ∼2.8-fold in muscle over the ∼20 y assessed. Mutation frequency in oocytes increased ∼2.5-fold until the age of 9 y, but did not increase after that, suggesting that oocytes of older animals maintain the quality of their mtDNA. We found the light-strand origin of replication (OriL) to be a hotspot for mutation accumulation with aging in liver. Indeed, the 33-nucleotide-long OriL harbored 12 variant hotspots, 10 of which likely disrupt its hairpin structure and affect replication efficiency. Moreover, in somatic tissues, protein-coding variants were subject to positive selection (potentially mitigating toxic effects of mitochondrial activity), the strength of which increased with the number of macaques harboring variants. Our work illuminates the origins and accumulation of somatic and germline mtDNA mutations with aging in primates and has implications for delayed reproduction in modern human societies.

Age-related accumulation of de novo mitochondrial mutations in mammalian oocytes and somatic tissues.

Mutations create genetic variation for other evolutionary forces to operate on and cause numerous genetic diseases. Nevertheless, how de novo mutations arise remains poorly understood. Progress in the area is hindered by the fact that error rates of conventional sequencing technologies (1 in 100 or 1,000 base pairs) are several orders of magnitude higher than de novo mutation rates (1 in 10,000,000 or 100,000,000 base pairs per generation). Moreover, previous analyses of germline de novo mutations examined pedigrees (and not germ cells) and thus were likely affected by selection. Here, we applied highly accurate duplex sequencing to detect low-frequency, de novo mutations in mitochondrial DNA (mtDNA) directly from oocytes and from somatic tissues (brain and muscle) of 36 mice from two independent pedigrees. We found mtDNA mutation frequencies 2- to 3-fold higher in 10-month-old than in 1-month-old mice, demonstrating mutation accumulation during the period of only 9 mo. Mutation frequencies and patterns differed between germline and somatic tissues and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. Additionally, we discovered a more pronounced genetic drift of mitochondrial genetic variants in the germline of older versus younger mice, arguing for mtDNA turnover during oocyte meiotic arrest. Our study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing directly in oocytes, which provided unprecedented resolution and minimized selection effects present in pedigree studies. Moreover, our work provides important information about the origins and accumulation of mutations with aging/maturation and has implications for delayed reproduction in modern human societies. Furthermore, the duplex sequencing method we optimized for single cells opens avenues for investigating low-frequency mutations in other studies.

Bottleneck and selection in the germline and maternal age influence transmission of mitochondrial DNA in human pedigrees.

Heteroplasmy-the presence of multiple mitochondrial DNA (mtDNA) haplotypes in an individual-can lead to numerous mitochondrial diseases. The presentation of such diseases depends on the frequency of the heteroplasmic variant in tissues, which, in turn, depends on the dynamics of mtDNA transmissions during germline and somatic development. Thus, understanding and predicting these dynamics between generations and within individuals is medically relevant. Here, we study patterns of heteroplasmy in 2 tissues from each of 345 humans in 96 multigenerational families, each with, at least, 2 siblings (a total of 249 mother-child transmissions). This experimental design has allowed us to estimate the timing of mtDNA mutations, drift, and selection with unprecedented precision. Our results are remarkably concordant between 2 complementary population-genetic approaches. We find evidence for a severe germline bottleneck (7-10 mtDNA segregating units) that occurs independently in different oocyte lineages from the same mother, while somatic bottlenecks are less severe. We demonstrate that divergence between mother and offspring increases with the mother's age at childbirth, likely due to continued drift of heteroplasmy frequencies in oocytes under meiotic arrest. We show that this period is also accompanied by mutation accumulation leading to more de novo mutations in children born to older mothers. We show that heteroplasmic variants at intermediate frequencies can segregate for many generations in the human population, despite the strong germline bottleneck. We show that selection acts during germline development to keep the frequency of putatively deleterious variants from rising. Our findings have important applications for clinical genetics and genetic counseling.

High Levels of Copy Number Variation of Ampliconic Genes across Major Human Y Haplogroups.

Because of its highly repetitive nature, the human male-specific Y chromosome remains understudied. It is important to investigate variation on the Y chromosome to understand its evolution and contribution to phenotypic variation, including infertility. Approximately 20% of the human Y chromosome consists of ampliconic regions which include nine multi-copy gene families. These gene families are expressed exclusively in testes and usually implicated in spermatogenesis. Here, to gain a better understanding of the role of the Y chromosome in human evolution and in determining sexually dimorphic traits, we studied ampliconic gene copy number variation in 100 males representing ten major Y haplogroups world-wide. Copy number was estimated with droplet digital PCR. In contrast to low nucleotide diversity observed on the Y in previous studies, here we show that ampliconic gene copy number diversity is very high. A total of 98 copy-number-based haplotypes were observed among 100 individuals, and haplotypes were sometimes shared by males from very different haplogroups, suggesting homoplasies. The resulting haplotypes did not cluster according to major Y haplogroups. Overall, only two gene families (RBMY and TSPY) showed significant differences in copy number among major Y haplogroups, and the haplogroup of a male could not be predicted based on his ampliconic gene copy numbers. Finally, we did not find significant correlations either between copy number variation and individual's height, or between the former and facial masculinity/femininity. Our results suggest rapid evolution of ampliconic gene copy numbers on the human Y, and we discuss its causes.

Preconception zinc deficiency disrupts postimplantation fetal and placental development in mice.

Zinc is an essential nutrient for optimal fertility, but the effects of preconception zinc deficiency on postimplantation development are not known. Female mice were fed a control or a zinc-deficient diet (ZDD) for 4-5 days before ovulation (preconception). Embryonic and/or placental development were evaluated on Days 3.5, 6.5, 10.5, 12.5, and 16.5 of pregnancy. The findings show a decrease in embryo length (31%, Day 10.5; 13%, Day 12.5; 10%, Day 16.5) and weight (23%, Day 16.5) in embryos from mothers fed a ZDD preconception. Zinc deficiency also caused a high incidence of pregnancy loss (46%, Day 10.5; 34%, Day 12.5; 51%, Day 16.5) compared to control (2%, Day 10.5; 7%, Day 12.5; 9%, Day 16.5). ZDD embryos transferred to normal recipients were 38% smaller and implantation rate was only 10% compared to 40% for controls. Trophoblast cell differentiation and implantation on Day 6.5 of pregnancy were compromised by preconception zinc deficiency. On Day 12.5 of pregnancy, placenta weight and area of fetal placenta were decreased 37% and 31%, respectively, by preconception zinc deficiency. Consistent with a smaller fetal placenta, expression of key placental transcripts, including Ar, Esx1, Syna, Tfeb, Dlx3, and Gcm1 mRNA, but not Ctsq mRNA, were decreased 30%-70% in the ZDD group. Preconception zinc deficiency caused 41%-57% of embryos to exhibit delayed or aberrant neural tube development, as examined by light microscopy and magnetic resonance imaging. Collectively, the findings provide evidence for the importance of preconception zinc in promoting optimal fertility and oocyte developmental potential.

Early avian follicular development is characterized by changes in transcripts involved in steroidogenesis, paracrine signaling and transcription.

The mechanisms associated with follicular activation and early growth are not well understood in avian species. Level of mRNA transcripts involved in steroidogenesis (STAR, HSD3B, CYP11A1, CYP19), paracrine signaling (AMH, BMP2, BMP4, BMP6, KITL, WNT4, and PCSK6) and transcription (SMAD1, SMAD2, SMAD3, SMAD5, SMAD9, FOXL3, FOXL2, NR5A1 (SF1), and WT1) were determined in small avian follicles 0.5, 1, and 2 mm in diameter after oocyte removal. STAR, HSD3B, CYP11A1, CYP19, PCSK6, FOXO3, and KITL mRNA increased 2- to 45-fold, while FOXL2, WT1, and WNT4 decreased 30-90% and NR5A1 did not change as follicles developed from 0.5 to 2 mm. Phosphorylated SMAD2, SMAD3, SMAD1/5/9 and FOXO3 proteins were found in both granulosa cells and oocytes of small (<0.5 mm) and larger (>1 mm) follicles. In contrast, non-phosphorylated FOXO3 protein was found in oocyte and granulosa cells of small follicles, but only in the oocyte of larger follicles. Culture of small avian follicles on the chorioallantoic membrane of chick embryos (in ovo) for 7 days caused changes in transcript levels that were similar to changes observed in vivo. The collective findings suggest that the growth of avian follicles from 0.5 to 2 mm is marked by an increase in steroidogenic capacity, and changes in paracrine signaling that may be important during early avian follicular development. Thus, a number of candidate marker genes were identified, and a method of follicle culture was developed to study early follicular development in a model avian species.

Perinatal nicotine exposure delays genital development in mice.

INTRODUCTION: Smoking has well-recognized endocrine disrupting effects in humans that have been linked specifically to nicotine in animal models. Nicotine's perinatal effects on genital development in mice, which is interpreted as a measure of endocrine disrupting activity, were evaluated in this report. METHODS: Pregnant C57B/6J dams were administered 50 microg/ml nicotine in drinking water from gestational day 9 until pups were weaned. Their pups' anogenital distances and weights were measured at birth and at weaning. A subset of the pups was weighed again in adulthood. RESULTS: Female and male mice had significantly reduced anogenital distance at birth; however, by 34 days of age, differences in anogenital distance were no longer apparent, while body weight, which had been equal at birth and weaning, was significantly reduced in mice exposed to perinatal nicotine. DISCUSSION: Perinatal nicotine exposure significantly delayed genital development and altered adolescent body weight in this model.

Activity-related behaviors in the hole-board predict nicotine consumption in C57B6 mice perinatally exposed to nicotine.

Hole-board behaviors of adolescent C57B/6 mice that had been exposed to nicotine during gestation and suckling were evaluated on postnatal days 34-36. Rearing on all three trials significantly predicted higher nicotine intake on a two-bottle choice test administered from days 37-42. For head pokes, there was a weak trend for lower head poking in the first trial to be predictive of higher nicotine intake. Locomotor activity only predicted higher nicotine consumption on the third trial. These results show that hole-board behaviors predict subsequent nicotine intake in mice exposed to nicotine perinatally, especially after habituation to the apparatus.

Optimizing DNA yield from buccal swabs in the elderly: attempts to promote buccal cell growth in culture.

Participation in genetic studies is often limited by a volunteer's reluctance to donate blood samples. We wished to determine if alternate, less painful, methods to venipuncture could be used to collect cells to provide DNA for genotyping, and whether the cells could be grown in culture for extraction of DNA. Volunteers in the study were comprised of two groups. Nine individuals from a university campus were recruited to provide samples for initial experiments. A second group of 710 twins and singletons from North Carolina and of African-American descent were a part of an ongoing study of age-related traits and participated in collection of buccal swabs via the mail. A protocol was generated that maximizes the recovery of DNA from buccal swabs, which are easier to handle than saline rinses. The DNA recovered is stable over several years, allowing genotype tests at a future date. Attempts to encourage growth of buccal epithelial cells recovered from swabs in tissue culture proved unsuccessful. Buccal swabs work well for the collection of DNA, especially from nonclinic-based volunteers, and can be sent via the mail to the laboratory for DNA extraction. Thus, an inexpensive and efficient method exists for genetic studies of population-based samples.