Jennifer R. Wood

MicroRNAs Are Absorbed in Biologically Meaningful Amounts from Nutritionally Relevant Doses of Cow Milk and Affect Gene Expression in Peripheral Blood Mononuclear Cells, HEK-293 Kidney Cell Cultures, and Mouse Livers

BACKGROUNDnMicroRNAs (miRNAs) regulate genes in animals and plants and can be synthesized endogenously. In milk, miRNAs are encapsulated in exosomes, thereby conferring protection against degradation and facilitating uptake by endocytosis. The majority of bovine miRNAs have nucleotide sequences complementary to human gene transcripts, suggesting that miRNAs in milk might regulate human genes.nnnOBJECTIVESnWe tested the hypotheses that humans absorb biologically meaningful amounts of miRNAs from nutritionally relevant doses of milk, milk-borne miRNAs regulate human gene expression, and mammals cannot compensate for dietary miRNA depletion by endogenous miRNA synthesis.nnnMETHODSnHealthy adults (3 men, 2 women; aged 26-49 y) consumed 0.25, 0.5, and 1.0 L of milk in a randomized crossover design. Gene expression studies and milk miRNA depletion studies were conducted in human cell cultures and mice, respectively. For comparison, feeding studies with plant miRNAs from broccoli were conducted in humans.nnnRESULTSnPostprandial concentration time curves suggest that meaningful amounts of miRNA (miR)-29b and miR-200c were absorbed; plasma concentrations of miR-1 did not change (negative control). The expression of runt-related transcription factor 2 (RUNX2), a known target of miR-29b, increased by 31% in blood mononuclear cells after milk consumption compared with baseline. When milk exosomes were added to cell culture media, mimicking postprandial concentrations of miR-29b and miR-200c, reporter gene activities significantly decreased by 44% and 17%, respectively, compared with vehicle controls in human embryonic kidney 293 cells. When C57BL/6J mice were fed a milk miRNA-depleted diet for 4 wk, plasma miR-29b concentrations were significantly decreased by 61% compared with miRNA-sufficient controls, i.e., endogenous synthesis did not compensate for dietary depletion. Broccoli sprout feeding studies were conducted as a control and elicited no detectable increase in Brassica-specific miRNAs.nnnCONCLUSIONnWe conclude that miRNAs in milk are bioactive food compounds that regulate human genes.

Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone†‡

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all‐trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA‐treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells. Mol. Reprod. Dev. 76: 11–21, 2009.

Genome-wide prediction of age at puberty and reproductive longevity in sows

Traditional selection for sow reproductive longevity is ineffective due to low heritability and late expression of the trait. Incorporation of DNA markers into selection programs is potentially a more practical approach for improving sow lifetime productivity. Using a resource population of crossbred gilts, we explored pleiotropic sources of variation that influence age at puberty and reproductive longevity. Of the traits recorded before breeding, only age at puberty significantly affected the probability that females would produce a first parity litter. The genetic variance explained by 1-Mb windows of the sow genome, compared across traits, uncovered regions that influence both age at puberty and lifetime number of parities. Allelic variants of SNPs located on SSC5 (27-28xa0Mb), SSC8 (36-37xa0Mb) and SSC12 (1.2-2xa0Mb) exhibited additive effects and were associated with both early expression of puberty and a greater than average number of lifetime parities. Combined analysis of these SNPs showed that an increase in the number of favorable alleles had positive impact on reproductive longevity, increasing number of parities by up to 1.36. The region located on SSC5 harbors non-synonymous alleles in the arginine vasopressin receptor 1A (AVPR1A) gene, a G-protein-coupled receptor associated with social and reproductive behaviors in voles and humans and a candidate for the observed effects. This region is characterized by high levels of linkage disequilibrium in different lines and could be exploited in marker-assisted selection programs across populations to increase sow reproductive longevity.

Genes involved in the immediate early response and epithelial–mesenchymal transition are regulated by adipocytokines in the female reproductive tract

Obesity increases the risk of female reproductive tract cancers, but the underlying mechanistic link between the two is ill‐defined. Thus, the objective of the current study was to identify obesity‐dependent changes in the expression of immediate early (IE) genes that contribute to cell proliferation and differentiation, and epithelial–mesenchymal transition (EMT) genes that promote cell migration. When HeLa cells were treated for 0–48u2009hr with IGF‐1, leptin, TNFα, or IL‐6, each individual adipocytokine altered the abundance of IE (cJUN, cFOS, and cMYC) and EMT (SNAI1, SNAI2, and TWIST1) mRNA abundance. For example, IGF‐1 increased cJUN and cFOS and decreased cMYC; leptin increased cFOS; IL‐6 increased cFOS and cMYC; and TNFα increased cJUN and cFOS mRNA abundance. Likewise, EMT gene expression was altered by IGF‐1, TNFα, and IL‐6. SNAI1 was increased by IGF‐1 and IL‐6; SNAI2 was increased by IGF‐1 and TNFα; and TWIST1 was increased by TNFα and IL‐6. Chronic exposure to adipocytokines also altered EMT gene expression in the whole uterus of obese compared to normal‐weight mice. Specifically, there was no difference in cJun, cFos, or cMyc mRNA abundance between normal‐weight and obese animals. Snai1, Snai2, and Twist1 mRNA abundance, however, was increased in the uterus of obese females and correlated with increased circulating IGF‐1 levels. These data indicate that obesity‐dependent alterations in adipocytokine levels regulate the expression of genes associated with cell proliferation and migration, and therefore may provide a plausible mechanism for obesity‐dependent increases in cancers of the female reproductive tract. Mol. Reprod. Dev. 79:128–137, 2012.

Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice

RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess.

Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; meanu200a=u200a102) and another with Low A4 (<20 ng/ml; meanu200a=u200a9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (Pu200a=u200a0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions.

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these four cell types. Analysis of the RNA present in each bovine cell type using Affymetrix microarrays yielded new cell-specific genetic markers, functional insight into the behavior of each cell type via Gene Ontology Annotations and Ingenuity Pathway Analysis, and evidence of small and large luteal cell lineages using Principle Component Analysis. Enriched expression of select genes for each cell type was validated by qPCR. This expression analysis offers insight into cell-specific behaviors and the differentiation process that transforms somatic follicular cells into luteal cells.

A polymorphism in myostatin influences puberty but not fertility in beef heifers, whereas µ-calpain affects first calf birth weight

The use of genetic markers to aid in selection decisions to improve carcass and growth characteristics is of great interest to the beef industry. However, it is important to examine potential antagonistic interactions with fertility in cows before widespread application of marker-assisted selection. The objective of the current experiment was to examine the influence of 2 commercially available markers currently in use for improving carcass traits, the myostatin (MSTN) F94L and μ-calpain (CAPN1) 316 and 4751 polymorphisms, on heifer development and reproductive performance. In Exp. 1, beef heifers (n = 146) were evaluated for growth and reproductive traits over a 3-yr period to determine if these polymorphisms influenced reproductive performance. In Exp. 2, heifers representing the 2 homozygous genotypes for the MSTN F94L polymorphism were slaughtered on d 4 of the estrous cycle and reproductive tracts were collected for morphological examination. In Exp. 1, there was a tendency (P = 0.06) for birth BW to be affected by MSTN with the Leu allele increasing birth BW in an additive fashion. Additionally, MSTN significantly affected the proportion of pubertal heifers by the start of the breeding season (P < 0.05) with the Leu allele additively decreasing the proportion pubertal; however, this did not result in a delay in conception or a decrease in pregnancy rates during the first breeding season (P > 0.15). The GT haplotype of CAPN1, which was previously associated with decreased meat tenderness, was associated with an additive decrease in birth BW of the first calf born to these heifers (P < 0.05). In Exp. 2, there were no differences between the MSTN genotypes for gross or histological morphology of the anterior pituitary, uterus, or ovaries (P > 0.05). From these results, we concluded that the MSTN F94L and CAPN1 polymorphisms can be used to improve carcass traits without compromising fertility in beef heifers. The influence of these markers on cow performance and herd life remains to be determined. While the delay in puberty associated with the MSTN F94L polymorphism did not negatively impact reproductive performance in heifers, caution should be used when combining this marker with other markers for growth or carcass traits until the potential interactions are more clearly understood.

Beef heifers with diminished numbers of antral follicles have decreased uterine protein concentrations

Previous research demonstrated a favorable relationship between the number of follicles detectable in the bovine ovary by ultrasonography and fertility, and bovine females with diminished numbers of antral follicles had smaller reproductive tracts. Therefore, we hypothesized that uterine function would be compromised in beef heifers with diminished numbers of antral follilcles. Angus heifers (n=480) were submitted for ultrasonographic evaluation of antral follicle number at 325 and 355d of age. After the second ultrasonographic examination, 40 pubertal heifers with the greatest average number of antral follicles (30.9±0.7) and 40 pubertal heifers with the lowest average number of antral follicles (14.2±0.7) were synchronized with two i.m. injections of prostaglandin F2α (25mg) administered 11d apart, and heifers were slaughtered on d6 (n=26 heifers/group) or d16 (n=14 heifers/group) of the resultant estrous cycle. The uterus was weighed, flushed for determination of protein content, and representative samples were fixed for determination of endometrial gland morphometry. Heifers in the Low group had fewer surface antral follicles and smaller reproductive tracts than heifers in the High group (P<0.01). Protein content of the uterine flushes was decreased in heifers in the Low group (P<0.01); however, there was no difference in the percent area of the endometrium occupied by endometrial glands. From these results, we conclude that the uterine environment of beef heifers with diminished numbers of antral follicles is less conducive to supporting early embryonic survival.

Progressive obesity alters the steroidogenic response to ovulatory stimulation and increases the abundance of mRNAs stored in the ovulated oocyte

Obese women who are able to attain pregnancy are at increased risk for early‐pregnancy loss due, in part, to reduced oocyte quality. We and others have demonstrated that female Lethal Yellow (LY) mice and female C57BL/6 mice fed a high fat diet (B6‐HFD) exhibit phenotypes consistent with human obesity. These studies also showed that zygotes collected from LY and B6‐HFD females have reduced developmental competence. The current hypothesis is that LY and B6‐HFD females exhibit an abnormal response to gonadotropin stimulation compared to C57BL/6 controls fed normal rodent chow (B6‐ND), resulting in the ovulation of oocytes with an altered molecular phenotype which may contribute to its reduced developmental competence. To test this hypothesis, age‐matched B6‐ND, B6‐HFD, and LY females were stimulated with exogenous gonadotropins, then circulating hormone levels and the phenotypes of ovulated oocytes were analyzed. There was no difference in ovulation rate or in the percentage of morphologically abnormal oocytes collected from the oviduct of any females. Progesterone and progesterone/estradiol ratios, however, were increased in B6‐HFD and LY compared to B6‐ND females 16u2009hr post‐human chorionic gonadotropin treatment. The transcript abundance of several candidate oocyte genes was also increased in B6‐HFD‐ and LY‐derived oocytes compared to B6‐ND‐derived oocytes. These data suggest that increased insulin and leptin levels of obese females elevated circulating progesterone concentrations, altered transcriptional activity during oocyte growth, and/or impaired mechanisms of RNA translation and degradation during oocyte maturation. These changes in mRNA abundance likely contribute to reduced oocyte quality and the subsequent poor embryogenesis associated with obesity. Mol. Reprod. Dev. 81: 735–747, 2014.