The synergetic process of folliculogenesis is mainly regulated by GDF-9 and BMP-15 as well as their receptors, such as BMPR2, TβR1 and BMPR1B. Expressions of these factors and the receptors are significant different among species. This study was designed to detect expression of GDF-9, BMP-15 and their receptors in mouse, porcine and human healthy follicles by immunohistochemistry. Three ages of human ovary were studied according to ovarian developmental schedule, i.e. gestational week (GW) 16, puberty (14 year-old) and adult (40 year-old). The results showed that both GDF-9 and BMP-15 were detectable in oocytes from primary follicles onward, besides, BMP-15 also presented in granulosa cells (GCs) and follicular follicle of mature follicles in mouse. However, they were maintained in oocytes and GCs from primordial to mature follicles in porcine except that GDF-9 was undetectable in GCs of mature follicles. For human ovary, GDF-9 presented in oocytes of primordial follicles in all samples, whereas BMP-15 was only observed in primordial follicle of adult ovary. Receptors, BMPR2, TβR1 and BMPR1B were found in oocytes and GCs of all follicles in mouse and porcine. In human, they were stained in oocytes from primordial follices but BMPR1B was not expressed in pubertal primordial follicles. Furthermore, we found that GDF-9, BMP-15 and three receptors distributed in adult corpus lutea. Collectively, our studies suggested that GDF-9, BMP-15 and their receptors might correlate with primordial follicular recruitment in pig and human. Positive expression of the receptors (BMPR2, TβR1 and BMPR1B)in primordial follicles of mouse ovaries indicated that these receptors might interact with others ligands besides GDF-9 and BMP-15 to regulate primordial follicular activity in mouse. Moreover, presence of GDF-9 in oocytes and BMP-15 in oocytes and GCs of mature follicles from mice and porcine elucidated coordinated roles of GDF-9 and BMP-15 in cumulus oophorus expansion. Additionally, expression of these factors in adult human corpus lutea suggested they play roles in corpus luteum activity.
Tibetan sheep is a unique breed living in Qinghai‐Tibet Plateau. Since MSTN/Smad signaling pathway plays a critical role in the regulation of muscle development, we aimed to study the mutton quality, mRNA expression of main transduction genes in the MSTN/Smad signaling pathway, and the effects of those genes on the mutton quality of Tibetan sheep in this study. Six‐month‐old Qinghai‐Tibetan sheep were selected, slaughtered, and their Longissimus lumborum, semitendinosus muscle, arm triceps, and quadriceps femoris muscle were collected. The mutton quality was evaluated, and gene expression and their association with the mutton quality were analyzed using RT‐qPCR. The results showed that the indexes of mutton quality were not significantly different between ewes and rams (p > .05) except for Warner–Bratzler shear force (WBSF) (p < .05). A total of 21 different fatty acids were detected in the muscles of Tibetan sheep, including nine types of SFA, four types of MUFA, and eight types of PUFA. The main transduction genes of the MSTN/Smad signaling pathway were found to be widely expressed in muscle tissues, but no significant differences were observed (p > .05). The correlation analysis of the main genes and mutton quality showed that MSTN was significantly correlated with redness and cooking time; Smad2, Smad3, Smad4, and TGFβRI had significant positive correlations with marbling in arm triceps; Smad3 and TGFβRII had strong negative correlations with pH24 h in Longissimus lumborum; Smad2 was negatively correlated with drip loss in Longissimus lumborum. In short, the expression level of MSTN in muscles was positively correlated with Smad2, Smad3, and Smad4 genes and negatively correlated with TGFβRII genes. Thus, the results of this study provide a theoretical basis for the regulation mechanism of the MSTN/Smad pathway on mutton quality.
SMAD4, SMAD5 and SMAD7 belonging to the transforming growth factor β (TGF-β) superfamily are indispensable for oocyte formation and development, ovarian organogenesis and folliculogenesis. However, only a few studies have investigated the characteristics of SMAD4, SMAD5 and SMAD7 in Tibetan sheep and the effect of their polymorphism on litter size. In this study, we examined the expression of SMAD4, SMAD5 and SMAD7 in 13 tissues of Tibetan sheep by reverse transcription-quantitative polymerase chain reaction. Further, cDNA of these genes was cloned, sequenced and subjected to bioinformatics analysis. DNA sequencing was also used to detect single nucleotide polymorphisms (SNPs). However, iM-LDRTM technology was used for SNP genotyping. Associations between polymorphisms and litter size were analyzed using data from genotyping of 433 Tibetan sheep. The results showed that the expression of SMAD4, SMAD5 and SMAD7 genes was ubiquitous in the tissues of Tibetan sheep, such as the ovary, uterus and oviduct, hypothalamus, hypophysis, heart, liver, spleen, lung, kidney, rumen, duodenum and longissimus dorsi. However, the expression was unbalanced and upregulated in the spleen, lung, ovary and uterus and downregulated in the longissimus dorsi. The bioinformatics analysis showed that SMAD4, SMAD5 and SMAD7 in Tibetan sheep encoded proteins of 533, 465 and 427 amino acids, respectively. Sequence homology analysis of the three proteins among other animals showed that the sequences of SMAD4, SMAD5 and SMAD7 of Tibetan sheep were similar to those in sheep, yak, cattle, dog, human, pig, chimpanzee, rhesus monkey and house mouse. Two synonymous mutations, g.51537A>G and g.319C>T, were detected in SMAD5 and SMAD7, respectively. The associations of these SNPs and litter size were determined, and it was found that both g.51537A>G and g.319C>T have no significant effect on the litter size of Tibetan sheep. The results provided novel insights into the molecular characterization, expression profiles and polymorphisms of SMAD4, SMAD5 and SMAD7 in Tibetan sheep, but our results do not support associations between these genes and the litter size of Tibetan sheep.
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