Genomic characterization of the world's longest selection experiment in mouse reveals the complexity of polygenic traits.

BACKGROUND: Long-term selection experiments are a powerful tool to understand the genetic background of complex traits. The longest of such experiments has been conducted in the Research Institute for Farm Animal Biology (FBN), generating extreme mouse lines with increased fertility, body mass, protein mass and endurance. For >140 generations, these lines have been maintained alongside an unselected control line, representing a valuable resource for understanding the genetic basis of polygenic traits. However, their history and genomes have not been reported in a comprehensive manner yet. Therefore, the aim of this study is to provide a summary of the breeding history and phenotypic traits of these lines along with their genomic characteristics. We further attempt to decipher the effects of the observed line-specific patterns of genetic variation on each of the selected traits. RESULTS: Over the course of >140 generations, selection on the control line has given rise to two extremely fertile lines (>20 pups per litter each), two giant growth lines (one lean, one obese) and one long-distance running line. Whole genome sequencing analysis on 25 animals per line revealed line-specific patterns of genetic variation among lines, as well as high levels of homozygosity within lines. This high degree of distinctiveness results from the combined effects of long-term continuous selection, genetic drift, population bottleneck and isolation. Detection of line-specific patterns of genetic differentiation and structural variation revealed multiple candidate genes behind the improvement of the selected traits. CONCLUSIONS: The genomes of the Dummerstorf trait-selected mouse lines display distinct patterns of genomic variation harbouring multiple trait-relevant genes. Low levels of within-line genetic diversity indicate that many of the beneficial alleles have arrived to fixation alongside with neutral alleles. This study represents the first step in deciphering the influence of selection and neutral evolutionary forces on the genomes of these extreme mouse lines and depicts the genetic complexity underlying polygenic traits.

Effects of different cyclooxygenase inhibitors on prostaglandin E2 production, steroidogenesis and ovulation of bovine preovulatory follicles.

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E2 (PGE2) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17ß-estradiol, progesterone, and prostaglandin E2 (PGE2) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixin-meglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE2 concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE2 is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.

Higher quality rather than superior quantity of oocytes determine the amount of fertilizable oocytes in two outbred Dummerstorf high-fertility mouse lines.

Dummerstorf fertility lines FL1 and FL2 represent two models of enhanced fertility characterized by the doubling of the litter size compared with an unselected control population (ctrl line, Dummerstorf FztDU). Both biodiverse FLs managed to reach this goal by increasing the ovulation rate per cycle, even showing decreased pregnancy rate and irregular oestrous cycle and metabolic hormone levels, compared with ctrl. The aim of the present study was to analyse oocytes in terms of quality and quantity by comparing the entire pool of oocytes per ovary, with those from the antral follicles within the same animal. We performed Brilliant Cresyl Blue staining as a non-invasive marker of oocyte quality in combination with an analysis of additional morphological indicators, e.g. cytoplasm clarity, cumulus cell layers, nuclear anatomy, size and shape. We compared our fertility lines with the unselected control population and with another independent line selected from the same founder population, showing lower litter size (DU6P). Our results suggest that fertility lines show decreased number of oocytes per ovary compared with DU6P but increased number of high-quality oocytes before ovulation. Hence, the raise in the ovulation rate and litter size of those super fertile mouse lines are not associated with an increased number of oocytes per ovary but rather with an increased number of higher quality fertilizable oocytes per cycle. In addition, the most conspicuous method to acquire oocytes with the highest quality in our lines is to assess their morphology, rather than their status after staining. All these discoveries together may be of fundamental importance for further studies in livestock farm animals showing some similar characteristics, e.g. irregular cycle or hormonal misbalances, to improve production while lowering costs, and in humans to increase the possibilities of successful pregnancies for couples undergoing in vitro fertilization (IVF).

Altered insulin, leptin and ghrelin hormone levels and atypical estrous cycle lengths in two highly fertile mouse lines.

Mouse models of decreased fertility mainly support scientific knowledge in the field of reproductive biology. In this study, we changed the perspective, using Dummerstorf high-fertility mouse lines FL1 and FL2 selected for increased reproductive performances that doubled the amount of ovulated oocytes per cycle and the number of offspring per litter compared to an unselected control line (founder population, FZTDU, ctrl). After recent observations, both fertility lines seem to show a lower pregnancy rate compared to ctrl together with an atypical reproductive cycle. We analysed the estrous cycle of those mice, but also plasma levels of insulin, glucagon, leptin and ghrelin that, when irregular, may have an impact on the reproductive cycle length by modifying the GnRH release. We included females of another independent line (DU6P), selected from the same founder population but independently of fertility traits, as an additional model of decreased pregnancy rate. We aim to evaluate if do they use a similar mechanism associated in the regulation of the estrous cycle or implicated in altered pregnancy mechanism compared to control, because they show a similar situation as FL2 line does, even without an increase in fertility parameters. We speculate that FLs' estrous cycle undergoes changes during the selection period and aim to demonstrate that some hormonal dysfunctions link with altered reproductive cycle, dampened pregnancy rates and reduced first delivery rates mostly in FL2, but also with higher-fertility phenotype rather than lower in both FLs.

Lower Plasmatic Levels of Saturated Fatty Acids and a Characteristic Fatty Acid Composition in the Ovary Could Contribute to the High-Fertility Phenotype in Dummerstorf Superfertile Mice.

In recent decades, fertility traits in humans as well as in farm animals have decreased worldwide. As such, it is imperative to know more about the genetics and physiology of increased or high fertility. However, most of the current animal models with reproductive phenotypes describe lower fertility or even infertility (around 99%). The "Dummerstorf high-fertility lines" (FL1 and FL2) are two unique mouse lines selected for higher reproductive performances, more specifically for higher number of pups per litter. We recently described how those superfertile mice managed to increase their reproductive phenotype by doubling the ovulation rate and consequently the litter size compared to the unselected mice of the same founder population. FLs show an unusual estrous cycle length and atypical levels of hormones that link reproduction and metabolism, such as insulin in FL1 and leptin in FL2. Moreover, we described that their higher ovulation rate is mostly due to a higher quality of their oocytes rather than their sheer quantity, as they are characterized by a higher quantity of high-quality oocytes in antral follicles, but the quantity of follicles per ovary is not dissimilar compared to the control. In the present study, we aimed to analyze the lipid composition of the fertility lines from plasma to the gonads, as they can connect the higher reproductive performances with their metabolic atypicalities. As such, we analyzed the fat content of FLs and fatty acid composition in plasma, liver, fat, oocytes of different quality, and granulosa cells. We demonstrated that those mice show higher body weight and increased body fat content, but at the same time, they manage to decrease the lipid content in the ovarian fat compared to the abdominal fat, which could contribute to explaining their ovarian quality. In addition, we illustrate the differences in fatty acid composition in those tissues, especially a lower level of saturated fatty acids in plasma and a different lipid microenvironment of the ovary. Our ongoing and future research may be informative for farm animal biology as well as human reproductive medicine, mostly with cases that present characteristics of lower fertility that could be reversed following the way-of-managing of Dummerstorf high-fertility lines.

Two mouse lines selected for large litter size display different lifetime fecundities.

We recently described two outbred mouse lines that were selected for large litter size at first delivery. However, lifetime fecundity appears to be economically more important for the husbandry of many polytocous species for which mouse lines might serve as bona fide animal models (e.g. for pigs). In the present study, we compared the lifetime fecundities of two highly fertile mouse lines (FL1 and FL2: >20 offspring/litter at first delivery) with those of an unselected control line (ctrl) and two lines that were selected for high body weight (DU6) and high protein mass (DU6P) without selection pressure on fertility. We tested the hypothesis that selection for large litter size at first parturition would also increase lifetime fecundity in mice, and we observed very large differences between lines. Whereas FL1 and ctrl delivered up to nine and ten litters, none of the DU6 and DU6P females gave birth to more than five litters. In line with this observation, FL1 delivered the most pups per lifetime (85.7/female). FL2 females produced the largest average litter sizes (20.4 pups/litter) in the first four litters; however, they displayed a reduced number of litters. With the exception of ctrl, litter sizes declined from litter to litter. Repeated delivery of litters with high offspring numbers did not affect the general health of FL females. The presented data demonstrate that two biodiverse, highly fertile mouse lines selected for large litter size at first delivery show different lifetime reproductive fitness levels. Thus, these mouse lines might serve as valuable mouse models for investigating lifetime productivity and longevity in farm animals.

Induction of cystic ovarian follicles (COFs) in cattle by using an intrafollicular injection of indomethacin.

The aim of this study was to establish a model to induce cystic ovarian follicles (COFs) in cattle using the cyclooxygenase inhibitor, indomethacin. Eighteen Holstein-Frisian cattle were synchronized with prostaglandin F2alpha (PGF2α) and gonadotropin-releasing hormone (GnRH). Ultrasound-guided transvaginal intrafollicular injections were performed in 23 preovulatory follicles with different concentrations of indomethacin 16 h after GnRH administration. An injection of 0.2 ml 35 µM indomethacin solution (resulting in a final concentration of 8 µg/ml in the follicular fluid) was the minimal dosage leading to COF formation. The induced COFs reached a maximum mean diameter of 36.9 ± 4.5 mm eleven days after injection. The estrous cycle was extended to 25-39 days. Luteinization was first observed 4 days after injection, accompanied by a slight increase in plasma progesterone concentration. The bioactivity of indomethacin was demonstrated by the decrease of prostaglandin E2 in the follicular fluid of three animals. The method presented here is minimally invasive and allows for the generation of defined COFs for further investigations.

Testicular transcriptional signatures associated with high fertility.

Factors of high fertility are poorly described. The majority of transgenic or knockout models with a reproductive phenotype are subfertile or infertile phenotypes. Few genotypes have been linked to improved reproductive performance (0.2%) or increased litter size (1%). In this study, we used a unique mouse model, fertility line FL1, selected for 'high fertility' for more than 170 generations. This strain has almost doubled the number of littermates as well as their total birth weight accompanied by an elevated ovulation rate and increased numbers of corpora lutea compared to a randomly mated and unselected control line (Ctrl). Here, we investigate whether the gonadal tissue of FL1 males are affected by 'co-evolution' after more than 40 years of female-focused selection. Using microarrays, we analysed the testicular transcriptome of the FL1 and Ctrl mice. These data were also compared with previously published female gonadal transcriptional alterations. We detected alterations in testicular gene expression, which are partly associated with female reproductive performance. Thus, female-focused selection for litter size has not only affected the female side, but also has been manifested in transcriptional alterations on the male gonadal organ. This suggests consequences for the entire mouse lines in the long run and emphasizes the perspective of inevitably considering both genders about mechanisms of high fertility.

Selection for female traits of high fertility affects male reproductive performance and alters the testicular transcriptional profile.

BACKGROUND: Many genes important for reproductive performance are shared by both sexes. However, fecundity indices are primarily based on female parameters such as litter size. We examined a fertility mouse line (FL2), which has a considerably increased number of offspring and a total litter weight of 180% compared to a randomly bred control line (Ctrl) after more than 170 generations of breeding. In the present study, we investigated whether there might be a parallel evolution in males after more than 40 years of breeding in this outbred mouse model. RESULTS: Males of the fertility mouse line FL2 showed reduced sperm motility performance in a 5 h thermal stress experiment and reduced birth rate in the outbred mouse line. Transcriptional analysis of the FL2 testis showed the differential expression of genes associated with steroid metabolic processes (Cyp1b1, Cyp19a1, Hsd3b6, and Cyp21a1) and female fecundity (Gdf9), accompanied by 150% elevated serum progesterone levels in the FL2 males. Cluster analysis revealed the downregulation of genes of the kallikrein-related peptidases (KLK) cluster located on chromosome 7 in addition to alterations in gene expression with serine peptidase activity, e.g., angiotensinogen (Agt), of the renin-angiotensin system essential for ovulation. Although a majority of functional annotations map to female reproduction and ovulation, these genes are differentially expressed in FL2 testis. CONCLUSIONS: These data indicate that selection for primary female traits of increased litter size not only affects sperm characteristics but also manifests as transcriptional alterations of the male side likely with direct long-term consequences for the reproductive performance of the mouse line.

Reproductive performance primarily depends on the female genotype in a two-factorial breeding experiment using high-fertility mouse lines.

Mouse models showing an improved fertility phenotype are barely described in the literature. In the present study, we further characterized two outbred mouse models that have been selected for the phenotype 'high fertility' for more than 177 generations (fertility lines (FL) 1 and 2). In order to delineate the impact of males and females on fertility parameters, we performed a two-factorial breeding experiment by mating males and females of the three different genotypes (FL1, FL2, unselected control (Ctrl)) in all 9 possible combinations. Reproductive performance, such as number of offspring per litter or total birth weight of the entire pup, mainly depends on the female genotype. Although the reproductive performance of FL1 and FL2 is very similar, their phenotypes differ. FL2 animals of both genders are larger compared to FL1 and control animals. Females of the control line delivered offspring earlier compared to FL1 and FL2 dams. Males of FL1 are the lightest and the only ones who gained weight during the two weeks mating period. To address whether this effect is correlated with differing serum androgen levels, we measured the concentrations of testosterone, dehydroepiandrosterone, 4-androstenedione, androstanediol and dihydrotestosterone in males of all three lines by GC-MS. We measured serum testosterone between 5.0 and 6.4 ng/mL, whereas the concentrations of the other androgens were at least one order of magnitude lower, with no significant differences between the lines. Our data indicate that reproductive outcome largely depends on the genotype of the female in a two-factorial breeding experiment and supports previous findings that the phenotype 'high fertility' is warranted by using different physiological strategies.

Effects of rutin and buckwheat seeds on energy metabolism and methane production in dairy cows.

Flavonoids are secondary plant metabolites with several health promoting effects. As dairy cows often suffer from metabolic imbalance and health problems, interest is growing in health improvements by plant substances such as flavonoids. Our group has recently shown that the flavonoids quercetin and rutin (a glucorhamnoside of quercetin) are bioavailable in cows when given via a duodenal fistula or orally, respectively, affect glucose metabolism, and have beneficial effects on liver health. Furthermore, flavonoids may reduce rumen methane production in vitro through their antibacterial properties. To test the hypothesis that rutin has effects on energy metabolism, methane production, and production performance in dairy cows, we fed rutin trihydrate at a dose of 100mg/kg of body weight to a group of 7 lactating dairy cows for 2 wk in a crossover design. In a second experiment, 2 cows were fed the same ration but were supplemented with buckwheat seeds (Fagopyrum tartaricum), providing rutin at a dose comparable to the first experiment. Two other cows receiving barley supplements were used as controls in a change-over mode. Blood samples were taken weekly and respiration measurements were performed at the end of each treatment. Supplementation of pure rutin, but not of rutin contained in buckwheat seeds, increased the plasma quercetin content. Methane production and milk yield and composition were not affected by rutin treatment in either form. Plasma glucose, ß-hydroxybutyrate, and albumin were increased by pure rutin treatment, indicating a possible metabolic effect of rutin on energy metabolism of dairy cows. In addition, we did not show that in vivo ruminal methane production was reduced by rutin. In conclusion, we could not confirm earlier reports on in vitro methane reduction by rutin supplementation in dairy cows in established lactation.

Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry.

The Effects of Oral Quercetin Supplementation on Splanchnic Glucose Metabolism in 1-Week-Old Calves Depend on Diet after Birth.

BACKGROUND: Inadequate colostrum supply results in insufficient intake of macronutrients and bioactive factors, thereby impairing gastrointestinal development and the maturation of glucose metabolism in neonatal calves. The flavonoid quercetin has been shown to have health-promoting properties, including effects in diabetic animals. However, quercetin interacts with intestinal glucose absorption and might therefore exert negative effects in neonates. OBJECTIVE: We evaluated the interaction between neonatal diet and quercetin feeding on splanchnic glucose metabolism in neonatal calves. METHODS: Calves (n = 28) were assigned to 4 groups and fed either colostrum or a milk-based formula on days 1 and 2 and supplemented daily with 148 µmol quercetin aglycone/kg body weight [colostrum with quercetin (CQ+)/formula with quercetin (FQ+)] or without this substance [colostrum without quercetin (CQ-)/formula with quercetin (FQ-)] from days 2-8. From day 3 onward, all calves received milk replacer. A xylose absorption test was performed on day 3, and on day 7, blood samples were collected to study glucose first-pass uptake after [(13)C6]-glucose feeding and intravenous [6,6-(2)H2]-glucose bolus injection. Plasma concentrations of metabolites and hormones were measured by taking additional blood samples. A biopsy specimen of the liver was harvested on day 8 to measure the mRNA expression of gluconeogenic enzymes. RESULTS: Higher postprandial plasma concentrations of glucose, lactate, urea, adrenaline, noradrenaline, insulin, and glucagon on day 7 in colostrum-fed calves indicate that metabolic processes were stimulated. Postabsorptive xylose and glucose plasma concentrations each increased by an additional 26%, and splanchnic glucose turnover decreased by 35% in colostrum-fed calves, suggesting improved glucose absorption and lower splanchnic glucose utilization in colostrum-fed calves. Quercetin supplementation resulted in higher noradrenaline concentrations and enhanced peak absorption and oxidation of [(13)C6]-glucose by 10%. Liver mitochondrial phosphoenolpyruvate carboxykinase mRNA abundance was reduced by 34% in colostrum-deprived calves. CONCLUSIONS: Feeding colostrum during the first 2 d of life is crucial for maturation of splanchnic glucose metabolism in calves. Supplementing quercetin improves gastrointestinal absorption capacity, particularly in colostrum-deprived calves.

Effects of a 6-wk intraduodenal supplementation with quercetin on energy metabolism and indicators of liver damage in periparturient dairy cows.

Periparturient dairy cows experience metabolic challenges that result in a negative energy balance (EB) and a range of postpartum health problems. To compensate for the negative EB, cows mobilize fatty acids from adipose tissues, which can lead to fatty liver disease, a periparturient metabolic disorder. Flavonoids, such as quercetin (Q), are polyphenolic substances found in all higher plants and have hepatoprotective potential and the ability to prevent or reduce lipid accumulation in the liver. In ruminants, few studies on the metabolic effects of Q are available, and thus this study was conducted to determine whether Q has beneficial effects on EB, lipid metabolism, and hepatoprotective effects in periparturient dairy cows. Quercetin was supplemented intraduodenally to circumvent Q degradation in the rumen. Cows (n=10) with duodenal fistulas were monitored for 7wk. Beginning 3wk before expected calving, 5 cows were treated with 100mg of quercetin dihydrate per kilogram of body weight daily in a 0.9% sodium chloride solution for a total period of 6wk, whereas the control cows received only the sodium chloride solution. The plasma flavonoid levels were higher in the Q-treated cows than in the control cows. A tendency for higher postpartum (pp) than antepartum (ap) plasma flavonoid levels was observed in the Q-treated cows than in the controls, which was potentially caused by a reduced capacity to metabolize Q. However, the metabolic status of the Q-treated cows did not differ from that of the control cows. The pp increases in plasma aspartate aminotransferase and glutamate dehydrogenase activities were less in the Q-treated cows than in the control cows. The Q had no effect on energy expenditures, but from ap to pp the cows had a slight decline in respiratory quotients. Irrespective of the treatment group, the oxidation of fat peaked after calving, suggesting that the increase occurred because of an increased supply of fatty acids from lipomobilization. In conclusion, supplementation with Q resulted in lower pp plasma aminotransferase and glutamate dehydrogenase, which indicated reduced liver damage. However, the direct effects of Q on the liver and the implications for animal performance remain to be investigated.

High-fertility phenotypes: two outbred mouse models exhibit substantially different molecular and physiological strategies warranting improved fertility.

Animal models are valuable tools in fertility research. Worldwide, there are more than 400 transgenic or knockout mouse models available showing a reproductive phenotype; almost all of them exhibit an infertile or at least subfertile phenotype. By contrast, animal models revealing an improved fertility phenotype are barely described. This article summarizes data on two outbred mouse models exhibiting a 'high-fertility' phenotype. These mouse lines were generated via selection over a time period of more than 40 years and 161 generations. During this selection period, the number of offspring per litter and the total birth weight of the entire litter nearly doubled. Concomitantly with the increased fertility phenotype, several endocrine parameters (e.g. serum testosterone concentrations in male animals), physiological parameters (e.g. body weight, accelerated puberty, and life expectancy), and behavioral parameters (e.g. behavior in an open field and endurance fitness on a treadmill) were altered. We demonstrate that the two independently bred high-fertility mouse lines warranted their improved fertility phenotype using different molecular and physiological strategies. The fertility lines display female- as well as male-specific characteristics. These genetically heterogeneous mouse models provide new insights into molecular and cellular mechanisms that enhance fertility. In view of decreasing fertility in men, these models will therefore be a precious information source for human reproductive medicine. Translated abstract A German translation of abstract is freely available at http://www.reproduction-online.org/content/147/4/427/suppl/DC1.

Low and high dietary protein:carbohydrate ratios during pregnancy affect materno-fetal glucose metabolism in pigs.

Inadequate dietary protein during pregnancy causes intrauterine growth retardation. Whether this is related to altered maternal and fetal glucose metabolism was examined in pregnant sows comparing a high-protein:low-carbohydrate diet (HP-LC; 30% protein, 39% carbohydrates) with a moderately low-protein:high-carbohydrate diet (LP-HC; 6.5% protein, 68% carbohydrates) and the isoenergetic standard diet (ST; 12.1% protein, 60% carbohydrates). During late pregnancy, maternal and umbilical glucose metabolism and fetal hepatic mRNA expression of gluconeogenic enzymes were examined. During an i.v. glucose tolerance test (IVGTT), the LP-HC-fed sows had lower insulin concentrations and area under the curve (AUC), and higher glucose:insulin ratios than the ST- and the HP-LC-fed sows (P < 0.05). Insulin sensitivity and glucose clearance were higher in the LP-HC sows compared with ST sows (P < 0.05). Glucagon concentrations during postabsorptive conditions and IVGTT, and glucose AUC during IVGTT, were higher in the HP-LC group compared with the other groups (P < 0.001). (13)C glucose oxidation was lower in the HP-LC sows than in the ST and LP-HC sows (P < 0.05). The HP-LC fetuses were lighter and had a higher brain:liver ratio than the ST group (P < 0.05). The umbilical arterial inositol concentration was greater in the HP-LC group (P < 0.05) and overall small fetuses (230-572 g) had higher values than medium and heavy fetuses (≥573 g) (P < 0.05). Placental lactate release was lower in the LP-HC group than in the ST group (P < 0.05). Fetal glucose extraction tended to be lower in the LP-HC group than in the ST group (P = 0.07). In the HP-LC and LP-HC fetuses, hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) was higher than in the ST fetuses (P < 0.05). In conclusion, the HP-LC and LP-HC sows adapted by reducing glucose turnover and oxidation and having higher glucose utilization, respectively. The HP-LC and LP-HC fetuses adapted via prematurely expressed hepatic gluconeogenic enzymes.

The Imitation of the Ovarian Fatty Acid Profile of Superfertile Dummerstorf Mouse Lines during IVM of Control Line Oocytes Could Influence Their Maturation Rates.

Declining human fertility worldwide is an attractive research target for the search for "high fertility" genes and pathways to counteract this problem. To study these genes and pathways for high fertility, the superfertile Dummerstorf mouse lines FL1 and FL2 are two unique model organisms representing an improved fertility phenotype. A direct reason for this remarkable characteristic of increased litter size, which reaches >20 pups/litter in both FLs, is the raised ovulation rate by approximately 100%, representing an impressive record in this field. Dummerstorf high-fertility lines incarnate extraordinary and singular models of high-fertility for other species, mostly farm animals, with the aim of improving production and reducing costs. Our main goal is to describe the genetic and molecular pathways to reach their phenotypical excellence, and to reproduce them using the control population. The large litter size and ovulation rate in Dummerstorf lines are mostly due to an increase in the quality of their oocytes, which receive a different intake of fat and are composed of different types and concentrations of fatty acids. As the follicular microenvironment plays a fundamental role during the oocytes development, in the present manuscript, we tried to improve the in vitro maturation technique by mimicking the fatty acid profile of FLs oocytes during the IVM of control oocytes. Currently, the optimization of the IVM system is fundamental mostly for prepubertal girls and oncological patients whose main source of gametes to restore fertility may be their maturation in vitro. Our data suggest that the specific fatty acid composition of FLs COCs can contribute to their high-fertility phenotype. Indeed, COCs from the control line matured in IVM-medium supplemented with C14:0 (high in FL2 COCs) or with C20:0, C21:0, C22:0, and C23:0 (high in FL1 COCs), but also control oocytes without cumulus, whose concentration in long-chain FAs are "naturally" higher, showing a slightly higher maturation rate. These findings represent an important starting point for the optimization of the IVM system using FA supplementation.

Alterations in gonadotropin, apoptotic and metabolic pathways in granulosa cells warrant superior fertility of the Dummerstorf high fertility mouse line 1.

The development and maturation of ovarian follicles is a complex and highly regulated process, which is essential for successful ovulation. During recent decades, several mouse models provided insights into the regulation of folliculogenesis. In contrast to the commonly used transgenic or knockout mouse models, the Dummerstorf high-fertility mouse line 1 (FL1) is a worldwide unique selection experiment for increased female reproductive performance and extraordinary high fertility. Interactions of cycle-related alterations of parameters of the hypothalamic pituitary gonadal axis and molecular factors in the ovary lead to improved follicular development and therefore increased ovulation rates in FL1 mice. FL1 females almost doubled the number of ovulated oocytes compared to the unselected control mouse line. To gain insights into the cellular mechanisms leading to the high fertility phenotype we used granulosa cells isolated from antral follicles for mRNA sequencing. Based on the results of the transcriptome analysis we additionally measured hormones and growth factors associated with follicular development to complement the picture of how the signaling pathways are regulated. While IGF1 levels are decreased in FL1 mice in estrus, we found no differences in insulin, prolactin and oxytocin levels in FL1 mice compared to the control line. The results of the mRNA sequencing approach revealed that the actions of insulin, prolactin and oxytocin are restricted local to the granulosa cells, since hormonal receptor expression is differentially regulated in FL1 mice. Additionally, numerous genes, which are involved in important gonadotropin, apoptotic and metabolic signaling pathways in granulosa cells, are differentially regulated in granulosa cells of FL1 mice.We showed that an overlap of different signaling pathways reflects the crosstalk between gonadotropin and growth factor signaling pathways, follicular atresia in FL1 mice is decreased due to improved granulosa cell survival and by improving the efficiency of intracellular signaling, glucose metabolism and signal transduction, FL1 mice have several advantages in reproductive performance and therefore increased the ovulation rate. Therefore, this worldwide unique high fertility model can provide new insights into different factors leading to improved follicular development and has the potential to improve our understanding of high fertility.

Functional cooperation between CREM and GCNF directs gene expression in haploid male germ cells.

Cellular differentiation and development of germ cells critically depend on a coordinated activation and repression of specific genes. The underlying regulation mechanisms, however, still lack a lot of understanding. Here, we describe that both the testis-specific transcriptional activator CREMtau (cAMP response element modulator tau) and the repressor GCNF (germ cell nuclear factor) have an overlapping binding site which alone is sufficient to direct cell type-specific expression in vivo in a heterologous promoter context. Expression of the transgene driven by the CREM/GCNF site is detectable in spermatids, but not in any somatic tissue or at any other stages during germ cell differentiation. CREMtau acts as an activator of gene transcription whereas GCNF suppresses this activity. Both factors compete for binding to the same DNA response element. Effective binding of CREM and GCNF highly depends on composition and epigenetic modification of the binding site. We also discovered that CREM and GCNF bind to each other via their DNA binding domains, indicating a complex interaction between the two factors. There are several testis-specific target genes that are regulated by CREM and GCNF in a reciprocal manner, showing a similar activation pattern as during spermatogenesis. Our data indicate that a single common binding site for CREM and GCNF is sufficient to specifically direct gene transcription in a tissue-, cell type- and differentiation-specific manner.