Effect of melatonin on ATG2B-mediated autophagy regulation in sheep granulosa cells with different FecB genotypes.

Melatonin (MLT) protects cells by reducing reactive oxygen species (ROS) levels, which are key for inducing cellular autophagy. The aim of this study was to investigate the molecular mechanisms underlying MLT regulation of autophagy in granulosa cells (GCs) with BMPR-1B homozygous (FecB BB) and wild type (FecB ++) mutations. GCs collected from small-tailed Han sheep with different FecB genotypes were typed using a TaqMan probe assay, and autophagy levels were found to be significantly higher in GCs with FecB BB than the levels in those with FecB ++. Autophagy-related 2 homolog B (ATG2B) was associated with cell autophagy and was highly expressed in GCs with the FecB BB genotype in small-tailed Han sheep. Overexpression of ATG2B in the GCs of sheep with both FecB genotypes promoted GC autophagy, and the contrary was observed after the inhibition of ATG2B expression. Subsequently, treatment of GCs with different genotypes of FecB and MLT revealed a significant decrease in cellular autophagy and an increase in ATG2B expression. Addition of MLT to GCs with inhibited ATG2B expression revealed that MLT could protect GCs by decreasing ROS levels, especially in GCs with FecB ++ genotype. In conclusion, this study determined that autophagy levels were significantly higher in sheep GCs with FecB BB genotype than the levels in those with FecB ++ genotype, which may have contributed to the difference in lambing numbers between the two FecB genotypes. Autophagy was regulated by ATG2B and was able to protect GCs by reducing the high levels of ROS produced following inhibition of ATG2B through the addition of MLT in vitro.

[Single base editing system mediates site-directed mutagenesis of genes GDF9 and FecB in Ouler Tibetan sheep].

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.

Poly cytosine (C)/poly adenine (A) modified probe for signal "on-off-on" assay of single-base mismatched dsDNA by a competitive mechanism.

Direct discrimination of single-base mismatched dsDNA by a simple method or strategy would provide enormous opportunities for applications in the fields of life sciences and disease diagnosis. Herein, the peroxidase-mimicking activity of a metal-organic framework nanoprobe (MOF) was well exploited for the direct discrimination of single-base mismatched dsDNA based on a competition-induced signal on-off-on mechanism. The single-base mismatched dsDNA related with FecB gene (usually guanine (G)/thymine (T) mismatch) and MIL-88B-NH2 were used as target and MOF model, respectively. Firstly, polyA/polyC were loosely adsorbed onto the MOFs via the weak interaction to block the peroxidase activity of MOF, inducing the signal transition from on to off. Unexpectedly, the single-base mismatched (GT) dsDNA could reverse the signal response of MOF probe from off to on. But it could not occur for other nonspecific mismatches, such as CT and TT-mismatched dsDNA. A synergistic interaction mechanism between multiple GT mismatches and polyA/polyC was attempted to explain the competitive dissociation of polyA/polyC from MOF for the recovery of peroxidase activity. With it, a wide linear detection ranges from 10-9 M-10-5 M of GT mismatched dsDNA and a low detection limit of 0.247 nM could be achieved, even in the real samples. The effect of mismatched base number or position was also studied. Such a simple, rapid, cost-effective, and one-step mixing and checking method for single-base mismatched dsDNA discrimination eliminates the complex sample pretreatment, special DNA probe design, exclusive amplification or signal readout means. It thus offers a simple and effective route for direct discrimination of mismatched dsDNA and might hold a huge potential for the applications in gene analysis, disease diagnosis, and elementary research in life sciences.

Newly reported 90-bp deletion within the ovine BMPRIB gene: Does it widely distribute, link to the famous FecB (p.Q249R) mutation, and affect litter size?

The study of the BMPRIB gene polymorphisms has become of great importance in sheep, because it provides critical genetic tools to improve reproductive efficiency in ewes. The purposes of this study were: to assess the genetic diversity of the 90-bp deletion polymorphism within the BMPRIB gene in 52 various sheep breeds; to examine its linkage to the p.Q249R variant in the same gene, as well as to analyze its association with litter size. Herein, a total of 2313 various sheep individuals were used to detect the presence of the 90-bp deletion, among them the six breeds (Australian White (AUW), Small-Tail Han (STH), Guiqian semi-fine wool (GQSFW) sheep, etc.) were genotyped using PCR-based genotyping technology (n = 1636), and 47 breeds worldwide (including STH sheep) were genotyped via whole genome sequencing (WGS) method (n = 677). Genotyping outcomes revealed that the polymorphism was segregated in 45 of 52 breeds with varying frequencies (0.05-0.93). It is pertinent to note that the 90-bp deletion is not linked to the famous p.Q249R SNP in population of interest, except in high prolific Hu sheep. The association analysis indicated that the Del-90-bp variant showed no significant association (P > 0.05) with litter size in AUW (n = 625), GQSFW (n = 75) and STH (n = 38) ewes. Nevertheless, further investigation is needed on the importance of the Del-90-bp variant as a DNA marker that could possibly serve as an additional selection criterion in breeding ewes with high productivity.

Are Copy Number Variations within the FecB Gene Significantly Associated with Morphometric Traits in Goats?

The Booroola fecundity (FecB) gene is a major fertility-related gene first identified in Booroola sheep. Numerous studies have investigated whether the FecB gene is a major fecundity gene in goats or whether there are other genes that play a critical role in goat fertility. Nevertheless, little attention has been paid to the role of the FecB gene in the body morphometric traits of goats, despite the positive relationship discerned between litter size and growth. We identified five copy number variations (CNVs) within the FecB gene in 641 goats, including 318 Shaanbei white cashmere (SBWC) goats, 203 Guizhou Heima (GZHM) goats, and 120 Nubian goats, which exhibited different distributions among these populations. Our results revealed that these five CNVs were significantly associated with goat morphometric traits (p < 0.05). The normal type of CNV3 was the dominant type and displayed superior phenotypes in both litter size and morphometric traits, making it an effective marker for goat breeding. Consequently, LD blocks in the region of 10 Mb upstream and downstream from FecB and potential transcription factors (TFs) that could bind with the CNVs were analyzed via bioinformatics. Although no significant LD block was detected, our results illustrated that these CNVs could bind to growth-related TFs and indirectly affect the growth development of the goats. We identified potential markers to promote litter size and growth, and we offer a theoretical foundation for further breeding work.

Effects of FecB Mutation on Estrus, Ovulation, and Endocrine Characteristics in Small Tail Han Sheep.

The Booroola fecundity gene (FecB) has a mutation that was found to increase the ovulation rate and litter size in Booroola Merino sheep. This mutation is also associated with the fecundity of small-tail han (STH) sheep, an important maternal breed used to produce hybrid offspring for mutton production in China. Previous research showed that the FecB gene affects reproduction in STH sheep, based on litter size records. However, the effects of this gene on estrus, ovulation, and endocrine characteristics in these sheep remain unclear. Here, we analyzed the traits mentioned earlier and compared them among the three FecB genotypes of STH ewes using estrus synchronization. Overall, 53 pluriparous ewes were selected from among 890 STH ewes and subjected to FecB genotyping for experiments to characterize estrous and ovulation rates. FecB heterozygous (+B) ewes presented an earlier onset of estrus (42.9 ± 2.2 h) and a shorter estrous cycle (17.2 ± 0.2 days) (P ≤ 0.05). The ovulation rates increased with the increasing copy number of the B allele (P ≤ 0.01). Ovulation time showed no significant differences among the three FecB genotypes. The serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone, estrogen (E2), and progesterone (P4) were measured in 19 of the ewes. Serum concentrations of E2 and FSH dramatically varied around the time of behavioral estrus. In FecB mutant homozygous (BB) ewes, E2 concentration had two peaks, which were higher (P ≤ 0.05) than those of ++ genotypes. FSH concentration of BB ewes was higher (P ≤ 0.05) than that of the ++ ewes just after estrus. The expression of the estrogen receptor 1 (ESR1) gene in the +B genotype was higher than in the other genotypes. Based on the data for the reproductive performance of STH ewes with the three FecB genotypes, our study suggests that the development of follicles in ewes with the B allele is dependent on the response to FSH regulated by E2 in the early stage. +B ewes, exhibiting moderate ovulation and litter size and a shorter estrous cycle, can be highly recommended in sheep crossbreeding systems for commercial mutton production. Moreover, this study provides useful information to conserve better and use the genetic resources of STH sheep in China.

Detection of mRNA Expression and Copy Number Variations Within the Goat Fec B Gene Associated With Litter Size.

The Booroola fecundity (Fec B ) gene, as the first major fecundity gene identified in Booroola sheep, has attracted careful attention. So far, previous research have uncovered the FecB mutation (Q249R) as the main mutation by virtue of which sheep exhibits multiple lambing phenomena. This mutation is now being intensively studied and widely used. However, such effect of the FecB mutation has not been applied to goats, and similar types of the Fec B gene in goats still need to be studied. Thus, the current study attempted to verify potential mutations in the goat Fec B gene as well as investigate their functions related to fecundity. First, Fec B expression was investigated in six different goat tissues, and we found that Fec B expression was highest in the mammary gland, followed by the ovary. Next, the influence of the Fec B gene was analyzed from a new perspective, where five potential copy number variations (CNVs) (CNV1-5) within the Fec B gene were identified for the first time, and then their effects on litter size were measured. Our results point out that CNV3 (P = 3.44E-4) and CNV5 (P = 0.034) could significantly influence the litter size of goats. Identically, the combination genotype of CNV3 and CNV5 which consisted of their dominant genotypes was also significantly associated with goat litter size (P = 7.80E-5). Hence, CNV3 and CNV5 could serve as potential DNA molecular markers applied to DNA editing and DNA microarray. Additionally, the abovementioned study has laid a theoretical foundation for the detection of potential fertility-related quantitative trait loci within the goat Fec B gene.

Polymorphism Detection of GDF9 Gene and Its Association with Litter Size in Luzhong Mutton Sheep (Ovis aries).

Litter size is one of the most important economic traits in sheep. GDF9 and BMPR1B are major genes affecting the litter size of sheep. In this study, the whole coding region of GDF9 was sequenced and all the SNPs (single nucleotide polymorphisms) were determined in Luzhong mutton ewes. The FecB mutation was genotyped using the Sequenom MassARRAY®SNP assay technology. Then, the association analyses between polymorphic loci of GDF9 gene, FecB, and litter size were performed using a general linear model procedure. The results showed that eight SNPs were detected in GDF9 of Luzhong mutton sheep, including one novel mutation (g.41769606 T > G). The g.41768501A > G, g.41768485 G > A in GDF9 and FecB were significantly associated with litter size in Luzhong mutton ewes. The g.41768485 G > A is a missense mutation in the mature GDF9 protein region and is predicted to affect the tertiary structure of the protein. The results preliminarily demonstrated that GDF9 was a major gene affecting the fecundity of Luzhong mutton sheep and the two loci g.41768501A > G and g.41768485 G > A may be potential genetic markers for improving litter size.

Effect of the Booroola fecundity (FecB) gene on the reproductive performance of ewes under assisted reproduction.

Reproductive traits are important factors in sheep production. The Booroola fecundity (FecB) gene-the first major gene for prolificacy identified in sheep-has a positive effect on ovulation rates and litter size under natural reproductive conditions. However, the effect of the FecB gene on reproductive performance under assisted reproduction, which uses many artificial hormones, remains unclear. In the present study, we evaluated the effect of FecB (BMPR-1B mutation) on reproductive performance under assisted reproduction, and examined offspring body weight at birth and weaning and survival rate at weaning. There were no differences among three genotype groups (homozygous carrier, BB; heterozygous carrier, B+; non-carrier, ++) in terms of estrus detection rate, time to estrus onset, or estrus duration following estrus synchronization (P > 0.05). The pregnancy rates at 60 d were similar among three genotype groups after artificial insemination (P > 0.05). However, the B allele had an additive effect on litter size (one copy resulted in an increase of 0.88 lambs and two copies produced an additional 0.41 lambs; P < 0.01), and increased lambing and fecundity rates (P < 0.01). After multiple ovulation, the average numbers of recovered embryos per ewe were 9.16 ±â€¯0.79, 8.20 ±â€¯0.77, and 8.44 ±â€¯0.61 in the BB, B+, and ++ ewes, respectively (P > 0.05). There were no differences in the fertilization rate or numbers of grade 1-2 embryos among different groups (P > 0.05). The birth and weaning weights of lambs from BB and B+ ewes were lower than those of lambs born from ++ ewes (P < 0.01) owing to the high fecundity. The survival rate of lambs at weaning did not differ among groups (P > 0.05). Our results indicated that the presence of the B allele had an additive effect on litter size after artificial insemination, but it did not influence the parameters of estrus synchronization and multiple ovulation. Furthermore, the higher prolificacy in ewes carrying the B allele was associated with a reduction in offspring body weight at birth and weaning.

Rapid visual detection of FecB gene expression in sheep.

Sheep play an important role in agricultural production and people's lives, and fecundity is one of the most important economic traits of sheep for sheep breeders. The Booroola fecundity (FecB) gene has a certain correlation with litter size in sheep. Therefore, this study aims to detect FecB expression quickly, accurately and visually. Here, we used the nucleic acid dye SYBR Green I to detect FecB with the amplification refractory mutation system (ARMS), which can efficiently, rapidly, economically and visually detect FecB expression in sheep. After ARMS polymerase chain reaction (PCR), SYBR Green I was directly added to the ARMS products, and whether the sheep carried FecB was judged by directly observing the color change in the PCR tube. Homozygous (BB) or heterozygous (B+) samples with FecB mutation were bright green, while wild type (++) samples without FecB were orange yellow. This study suggested that this method has 100% accuracy and 0.5 ng/µL sensitivity. To our knowledge, this is the first report that shows the integration of the ARMS with SYBR Green I to detect FecB expression in sheep visually.