Comparison of embryo quality and pregnancy outcomes for patients with low ovarian reserve in natural cycles and mildly stimulated cycles: a cohort study.

BACKGROUND: As women with low ovarian reserve embark on the challenging journey of in-vitro fertilisation (IVF) treatment, the choice between natural and mildly stimulated cycles becomes a pivotal consideration. It is unclear which of these two regimens is superior for women with low ovarian reserve. Our study aims to assess the impact of natural cycles on embryo quality and pregnancy outcomes in women with low ovarian reserve undergoing IVF treatment compared to mildly stimulated cycles. METHODS: This retrospective study enrolled consecutive patients with low ovarian reserve who underwent IVF/intracytoplasmic sperm injection (ICSI) at Guangdong Second Provincial General Hospital between January 2017 and April 2021. The primary outcome for pregnancy rate of 478 natural cycles and 448 mild stimulated cycles was compared. Secondary outcomes included embryo quality and oocyte retrieval time of natural cycles. RESULTS: The pregnancy rate in the natural cycle group was significantly higher than that in the mildly stimulated cycle group (51.8% vs. 40.1%, p = 0.046). Moreover, natural cycles exhibited higher rates of available embryos (84.1% vs. 78.6%, p = 0.040), high-quality embryos (61.8% vs. 53.2%, p = 0.008), and utilisation of oocytes (73% vs. 65%, p = 0.001) compared to mildly stimulated cycles. Oocyte retrievals in natural cycles were predominantly performed between 7:00 and 19:00, with 94.9% occurring during this time frame. In natural cycles with high-quality embryos, 96.4% of oocyte retrievals were also conducted between 7:00 and 19:00. CONCLUSION: Natural cycles with appropriately timed oocyte retrieval may present a valuable option for patients with low ovarian reserve.

In the realm of in-vitro fertilisation (IVF) treatment, women with low ovarian reserve often face the crucial decision of opting for natural or mildly stimulated cycles. This retrospective study, conducted between January 2017 and April 2021 at Guangdong Second Provincial General Hospital, delves into the impact of these cycles on pregnancy outcomes. Examining 478 natural cycles and 448 mildly stimulated cycles, the study reveals a notably higher pregnancy rate in the natural cycle group (51.8% vs. 40.1%). Additionally, natural cycles demonstrated higher rates of available embryos, high-quality embryos, and oocyte utilisation compared to their mildly stimulated counterparts. The findings suggest that natural cycles, with proper oocyte retrieval timing, could be a favourable choice for those with low ovarian reserve seeking IVF treatment.

Continuous light exposure influences luteinization and luteal function of ovary in ICR mice.

With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.

Investigation of the association between goat DNMT3B gene polymorphism and growth traits.

The DNA methyltransferase 3 beta (DNMT3B) gene is key for DNA methylation and has been well recognized in regulating growth and development. A previous observation indicated that an 11-bp indel of DNMT3B affected the reproductive traits in goats, yet the effect of this polymorphism on body measurement traits in goats has not been reported. This study aims to investigate the associations between DNMT3B gene polymorphism and goat growth traits. We investigated this 11-bp indel in 2184 goats and three genotypes have been found in Shaanbei white cashmere goat (SBWC): insertion/insertion (II), deletion/deletion (DD) and insertion/deletion (ID). Only ID and DD genotypes were detected in Nubian goats and Guizhou heima goat (GZHM). The allele frequencies analyzed revealed that the 'D' allele frequencies were higher in all three goat breeds. Further association analysis demonstrated that this indel is markedly associated with the cannon circumference (CC) and cannon circumference index (CCI) of SBWC and cannon circumference (CC) of Nubian goats (p < .05). The CC and CCI are essential indicators to measure the growth status of goats. In summary, our study sheds some light on the potential impact of the 11-bp indel polymorphism of the DNMT3B gene on improving the growth traits in goats.

Goat CLSTN2 gene: tissue expression profile, genetic variation, and its associations with litter size.

Calsyntenin-2 (CLSTN2) is involved in cell proliferation, differentiation, cell death, tumorigenesis, and follicular expression. Although CLSTN2 has been identified as a potential candidate gene for sheep prolificacy, no studies have been done on its effect on goat prolificacy. The purpose of this study was to identify mRNA expression and genetic variation within goat CLSTN2, and its association with prolificacy. Herein, we uncovered significant differences in mRNA levels of the CLSTN2 gene in different tissues in female goats (p < 0.01), including ovary tissue. Nine putative indels were designed to investigate their correlation to litter size, but only one 16-bp deletion was discovered in female Shaanbei white cashmere goats (n = 902). We discovered that a 16-bp deletion within the CLSTN2 gene was significantly correlated with first-born litter size (p = 0.0001). As shown by the chi-squared test, the genotypic II of single-lambs and multi-lambs was dramatically higher than with genotype ID (p = 0.005). Our findings suggest that indel within the CLSTN2 gene is a candidate gene affecting prolificacy in goats and may be used for Marker Assisted Selection (MAS) in goats.

A prospective cohort study of the relationship between the withdrawal time and the detection rate of colorectal adenoma.

BACKGROUND AND AIMS: The effect of colonoscopy withdrawal time (WT) beyond 6 min on colorectal adenoma detection rate (ADR) is unclear. We focused on the relationship between WT and ADR. MATERIALS AND METHODS: This study was a prospective observational study involving 437 patients who underwent colonoscopy at Tongren Hospital in Shanghai from 1 July 2020 to 31 August 2020. Patients were divided into two groups according to whether the WT was >6 min. Age, sex, body mass index (BMI), defoaming rate score, Boston bowel preparation scale (BBPS), primary colonoscopy, hypertension, diabetes mellitus, dietary preparation 1 day before the examination, and abdominal surgery history factors were analysed by univariate and multivariable logistic regression to explore the odds ratios (ORs) of ADR in two WT groups. Restricted cubic spline regression was used to further analyze the relationship between WT and the ORs of adenoma detection. RESULTS: The ADR among 437 patients was 17.16% (75/437). Multivariable regression analysis showed that in the group with WT >6 min, patients aged ≥50 years old and male could have an increased risk of adenoma detection (OR 5.80, 95% CI 2.32-14.47; p < .001; OR 2.30, 95% CI 1.19-4.43; p = .013). The cubic spline curve showed that the ADR increased with time for WT of 6-8 min, and the highest ADR was achieved when the WT was controlled at 8 min (WT = 5.997, OR = 0.997; WT = 8.240 min, OR = 3.092). CONCLUSION: The highest ADR was achieved when the WT of colonoscopy was controlled at 8 min.

The 12-bp indel in the SMAD family member 2 gene is associated with goat growth traits.

SMAD family member 2 (SMAD2) is a member of the TGFß signaling pathway and functions as an essential regulator in the processes of development, cell proliferation, and bone formation. A previous observation reported that a 12-bp deletion of this gene affected the litter size in goats. However, according to our knowledge, no study has reported an association between this polymorphism and goat body measurement traits. The purpose of this study was to investigate the association of the insertion/deletion (indel) within the SMAD2 gene with the growth traits of goats. The indel polymorphism was found to be significantly associated with chest width and bust (p < 0.05), while cannon circumference was significantly the strongest compared to other traits (p < 0.01) and individuals with the DD genotypes were more dominant genotypes than other genotypes. In summary, we found evidence that the 12-bp indel within the SMAD2 gene could improve goat body measurement traits, paving the way for marker-assisted selection in the field of goat genetics and breeding.

Polymorphism within the GATA binding protein 4 gene is significantly associated with goat litter size.

GATA binding protein 4 (GATA4) is a typical transcription binding factor, and its main functions include regulating the proliferation, differentiation and apoptosis of ovarian granulosa cells, promoting spermatogenesis and sex differentiation, implying that this gene have possibly roles in animal reproduction. This study aims to detect five potential insertion/deletions (indels) of the GATA4 gene in 606 healthy unrelated Shaanbei white cashmere (SBWC) goats and analyze its association with the litter size. The electrophoresis and DNA sequencing identified two polymorphic indels (e.g., P4-Del-8bp and P5-Ins-9bp indel). Then T-test analysis showed that P4-Del-8bp was significantly correlated with litter size (p = 0.022) because of two different genotypes detected, e.g., insertion-deletion (ID) and deletion-deletion (DD), and the average litter size of individuals with DD genotype goats was higher than that of others. However, there was no correlation between P5-Ins-9bp and lambing of goats. Chi-square (X2) test found that the distribution of and P4-Del-8bp genotypes (X2 = 6.475, p = 0.011) was significantly different between single and multiple-lamb groups, while P5-Ins-9bp (X2 = 0.030, p = 0.862) was not. Therefore, these findings revealed that P4-Del-8bp polymorphism of goat GATA4 gene was a potential molecular marker significantly associated with litter size, which can be used for the marker-assisted selection (MAS) breeding to improve goat industry.

Detection of small sequence variations within the goat GHR gene and its effects on growth traits.

Growth hormone receptor (GHR) gene is considered to be an important candidate gene in growth traits. Therefore, the purpose of this study was to detect whether there were potential indel variations in the GHR gene that were related to the growth traits of the Shaanbei white cashmere goats (SBWC). In this study, genomic DNA from 931 healthy SBWC individuals were used to verify the relationship between the indel of the GHR gene and growth traits. Two indel variants, P49-bp indel in intron 1 and P1410-bp indel in 3'-UTR, were confirmed. Association analyses demonstrated that these two indel polymorphism loci were associated with the chest circumference and chest width of SBWC. Additionally, for the P49-bp and P1410-bp indel loci, the ID and II genotypes were dominant genotypes, respectively. Moreover, the genotypic distributions of these two indel loci in SBWC were significantly different from those in three other Chinese indigenous goat breeds (HNBG, GZDG and IMWC) (p < 0.05). Taken together, two indel loci (P49-bp indel and P1410-bp indel) both significantly affected the growth traits of goats. This illustrated that these two indel loci might be the potential DNA marker for use in improving the selection and breeding of goats.

PAX7 promotes CD49f-positive dairy goat spermatogonial stem cells' self-renewal.

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs.

Single-cell RNA sequencing analysis of mouse follicular somatic cells†.

Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell-cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell-cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.

Deletion mutation within the goat PPP3CA gene identified by GWAS significantly affects litter size.

The protein phosphatase 3 catalytic subunit α (PPP3CA) gene is a high reproduction traits candidate gene for goats as revealed by a genome-wide association study. The aim of this work was to explore the genetic variations of the goat PPP3CA as well as to evaluate the genetic effects on litter size. Three novel insertions/deletions (indels) within the goat PPP3CA were found and their minor allelic frequencies (MAF) were 0.105, 0.066, and 0.042, respectively. The results showed that only the 20bp indel polymorphism was significantly associated with litter size in Shaanbei white cashmere goats (P<0.05) and individuals with deletion/deletion (DD) genotypes demonstrated the junior phenotypes when compared with those with other genotypes. These findings suggested that the 20bp indel is a potential DNA marker for selecting superior individuals in marker-assisted selection for breeding concerning fecundity in goats.

Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes.

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

Two indel variants of prolactin receptor (PRLR) gene are associated with growth traits in goat.

Prolactin receptor (PRLR) gene plays a crucial role in the milk production, reproduction and the growth of mammals. To fully characterize the structure of the mutation and to further study the function of the goat PRLR gene, two insertion/deletion (indel) loci (12 bp; 16-bp; 5-bp) were detected in 1038 Shaanbei white cashmere (SBWC) goats. Associated analysis revealed that the 16-bp indel mutation was significantly associated the body length, body height, chest depth (CD), body length index (BLI), heart girth index and cannon circumference index (CCI) (p < 0.05). The polymorphism of 5-bp indel was significantly associated with CD, heart girth, CCI and BLI (p < 0.05). Overall, individuals with genotype DD showed better phenotypic traits than individuals with other genotypes at the two loci of PRLR gene in SBWC goat. These findings suggested that the two novel indels within the caprine PRLR gene could be considered as effective DNA molecular markers and could provide a valuable theoretical basis for the application of marker-assisted selection in the goat industry.

A deletion mutation within the ATBF1 gene is strongly associated with goat litter size.

AT motif-binding factor (ATBF1) is a transcription factor that suppresses carcinogenesis and induces neuronal differentiation, as well as it directly interacts with estrogen receptor (ER) and regulates progesterone receptors signaling. Thus ATBF1 gene will possibly greatly affect reproductive tratis in animals. According to our available findings, two indel (insertion/deletion) variants (12-bp indel; 6-bp indel) within the ATBF1 gene were found in Shaanbei White cashmere goats (SWCG). The objective of this work was to investigate their association with litter size in Shaanbei White Cashmere goat. The association analyses uncovered that ATBF1-12bp indel was strongly related to the first-birth litter size in SWCG population (n = 1195, P = 1.0 × 10-3). Individuals with DD genotype (n = 445) had the best litter size when compared with those of ID genotype (n = 420) and DD genotype (n = 330). Besides, Chi-square tests of different litter size individuals showed that multi-kids individuals had higher "D" allelic frequencies and "DD" genotypic frequencies (p < 0.01). These findings suggested the ATBF1-12bp deletion mutation was significantly associated with reproduction-related trait, suggesting that this deletion could be used as an effective molecular marker for litter size in goat breeding.

p53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly-L-lysine.

Male germline stem cells (mGSCs) can transmit genetic materials to the next generation and dedifferentiate into pluripotent stem cells. However, in livestock, mGSC lines are difficult to establish, because of the factors that affect their isolation and culture. The extracellular matrix serves as a substrate for attachment and affects the fate of these stem cells. Poly-L-lysine (PL), an extracellular matrix of choice, inhibits and/or kills cancer cells, and promotes the attachment of stem cells in culture. However, how it affects the characteristics and potentials of these stem cells in culture needs to be elucidated. Here, we isolated, enriched and cultured dairy goat mGSCs on five types of extracellular matrices. To explore the best extracellular matrix to use for culturing them, the characteristics and proliferation ability of the cells were determined. Results showed that the cells shared several characteristics with previously reported mGSCs, including the poor effect of PL on their proliferative and colony-forming abilities. Further examination showed upregulation of p53 expression in these cells, which could be inhibiting their proliferation. When a p53 inhibitor was included in the culture medium, it was confirmed to be responsible for the inhibition of proliferation in mGSCs. Optimal concentration of the inhibitor in the culture of these cells was 5 µM. Furthermore, addition of the p53 inhibitor increased the expression of the markers of self-renewal and cell cycle in goat mGSCs. In summary, suppressing p53 is beneficial for the proliferation of dairy goat mGSCs, cultured on PL.

The N-terminal His-tag affects the triglyceride lipase activity of hormone-sensitive lipase in testis.

The sterility of hormone-sensitive lipase (HSL) knockout mice clearly shows the link between lipid metabolism and spermatogenesis. However, which substrate or product of this multifunctional lipase affects spermatogenesis is unclear. We found that an HSL protein with a His-tag at the N-terminus preserved the normal hydrolase activity of cholesteryl ester (CE) but the triglyceride lipase (TG) activity significantly decreased in vitro. Therefore, mice with this functionally incomplete HSL (His-HSL) were produced on a background of HSL deficiency (HSL-/- h). As a result, HSL-/- h testis has an 8.65-fold higher CE activity than wild-type testis but a twofold higher TG activity than wild-type testis. To compare His-HSL and wild-type HSL in vitro and in vivo, we confirmed that the His-tag significantly suppressed HSL TG activity. From our results, we believe that TG activity was affected by the His-tag insertion, but CE activity was not influenced. Furthermore, the His-tag protected HSL from binding to the inhibitor BAY. From our study, TG activity and BAY binding sites were affected by N-terminal His-tag insertion.

High-throughput paper spray mass spectrometry via induced voltage.

RATIONALE: Paper spray (PS) has been developed as a method of choice for point-of-care analysis in many real cases, where its applications can be further expanded with delicate high-throughput design. To achieve this goal, we developed a new PS regime, with the assembly of an induced high voltage into the ion source. Compared with regular DC high voltage, the newly developed setup is capable of high-throughput, simple configuration and rapid switching between individual papers without complicated electric/mechanic design. METHODS: A device of high-throughput induced PS (IPS) was designed by using a two-dimensional (2D) rotating platform equipped with a circular glass plate. The paper substrate was placed on the circular glass plate and separated from the electrode. The method avoids physical contact between the electrode and the sample. Charged droplets were generated at the paper tip once an induced high voltage was applied to a wet paper. RESULTS: A relatively rapid analytical speed of 2.6 s per sample was achieved via IPS-MS. Rapid quantification of amitriptyline (AMT) in complicated matrices was obtained within 1 min using an isotope internal standard method. Limits of detection for AMt in urine, FBS and blood were calculated to be 1.04, 0.84 and 1.33 ng/mL, respectively. In addition, high-throughput IPS-MS can be used for chemical reaction monitoring. CONCLUSIONS: We have demonstrated the versatility of high-throughput IPS-MS in ambient ionization, which successfully simplified the experimental installation and facilitated the experimental operation. Therefore, we believe that high-throughput IPS-MS analysis will be widely used for discovering drugs and screening reactions, and the present design has the potential for applications in paper chip-MS analysis.

Lycopene and alpha-lipoic acid improve semen antioxidant enzymes activity and cashmere goat sperm function after cryopreservation.

Cashmere goats, a unique biological resource in China, have the highest cashmere yield and best fiber quality in the world. The goal of this study was to determine the effects of cryopreserving with lycopene (LP) and alpha-lipoic acid (ALA) on the physiological characteristics of Cashmere goat spermatozoa. The results showed that sperm motility, acrosome integrity, membrane integrity, and mitochondrial activity of goat spermatozoa were greater in extenders containing 1.0 mg/mL LP and 10 µg/mL ALA (P < 0.05). Furthermore, higher SOD, CAT, and GSH-Px levels in semen occurred for extenders with 1.0 mg/mL LP and 10 µg/mL ALA compared with that of other treatments and the control group (P < 0.05). Interestingly, this study also combined LP + ALA in the extender. The results showed that the sperm motility, membrane integrity, and acrosome integrity in 1.0 mg/mL + 5 µg/mL in the LP + ALA group were significantly elevated compared with that of the control group (P < 0.05). Moreover, 1.0 mg/mL + 5 µg/mL LP + ALA addition increased SOD, CAT, and GSH-Px in spermatozoa more than 1.0 mg/mL LP and 10 µg/mL ALA (P < 0.05). In addition, the results of AI showed that the pregnancy rates were higher in the 1.0 mg/mL + 5 µg/mL LP + ALA group than the control group (P < 0.05). In conclusion, the addition of LP + ALA to extender solutions protects goat spermatozoa from ROS attack by improving antioxidant enzymes activity, and the results suggested that freezing extenders supplemented with 1.0 mg/mL + 5 µg/mL LP + ALA would be beneficial to the Cashmere goat breeding industry.

MiR-302 enhances the viability and stemness of male germline stem cells.

MicroRNAs were reported to be able to regulate mGSCs' self-renewal through post-transcriptional inhibition of gene expression. miR-302 worked as one important microRNA family existed mainly in human ESCs, and its role in mGSCs has not been reported yet. To elucidate the role of miR-302 in dairy goat mGSCs, the expression profile of miR-302 was explored through qPCR and FISH. Furthermore, to detect the function of miR-302, the expression vector containing miR-302 was transfected into mGSCs, and then, the cell cycle, the cell apoptosis and the genes associated with mGSCs' self-renewal and differentiation were examined. The results showed that miR-302 expressed in testis moderately and located on the basement of seminiferous tubes which shared the same location as mGSCs. Transfection of the vector containing miR-302 fragment into the immortalized mGSCs obviously enhanced the cell proliferation ability and the attachment ability, also, promoted the expression level of CD49f and OCT4. Also, miR-302 reduced the cell apoptosis and downregulated the expression of P21. miR-302 sustained mGSCs' proliferation in vitro.

Tß4-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics.

Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tß4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. To obtain genetically modified cells as nuclear donors, we co-transfected donor vectors into fetal fibroblasts of cashmere goats. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Via determination of the copy numbers and integration sites, the Tß4 gene was successfully inserted into the goat genome. Histological examination of skin tissue revealed that Tß4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tß4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). Our results indicate that Tß4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.