Semen Protein CRISP3 Promotes Reproductive Performance of Boars through Immunomodulation.

Semen proteins play an important role in male reproductive performance and sperm fertilization ability and can be used as potential biomarkers to evaluate male fertility. The role of cysteine-rich secretory protein 3 (CRISP3) in male reproduction remains unknown. This study aimed to investigate the role of CRISP3 in the reproductive performance of boars. Our results showed that the CRISP3 protein content was significantly and positively correlated with boar fertility, sow delivery rate, and litter size. CRISP3 is highly expressed in the bulbourethral gland of adult boars and is enriched in the seminal plasma. It is localized in the post-acrosomal region of the sperm head and migrates to the anterior end of the tail after capacitation. The CRISP3 recombinant protein did not affect sperm motility and cleavage rate, but it significantly downregulated the mRNA expression of inflammatory factors IL-α, IL-1ß, and IL-6 and the protein expression of IL-α and IL-6 in lipopolysaccharide (LPS)-induced RAW264.7 cells, indicating that CRISP3 has an immunomodulatory function. In conclusion, our study suggests that semen CRISP3 protein levels positively correlate with reproductive performance, which may be achieved by regulating immune responses in the female reproductive tract.

Cloprostenol sodium improves reproductive performance of multiparous sows during lactation.

This study aimed to determine the effect of prostaglandin F2α (PGF2α) analog (D-cloprostenol sodium and DL-cloprostenol sodium) administration on the milk yield of multiparous sows (MS) and piglet growth performance. In total, 320 Landrace×Yorkshire parturient MS were randomly divided into three groups on day 115 of pregnancy: without treatment (N = 50), with 75 µg D-cloprostenol sodium (N = 137), and with 200 µg DL-cloprostenol sodium (N = 133). After delivery, the sows treated with D-cloprostenol sodium and DL-cloprostenol sodium were randomly allocated into three subgroups, respectively: (i) no additional treatment after farrowing; (ii) administration of cloprostenol sodium at 3 h and 5 days after farrowing; and (iii) administration of cloprostenol sodium at 3 h, 5 days, and 10 days after farrowing. Cloprostenol sodium effectively induced sows to synchronize parturition approximately 23 h after administration and increased the daytime delivery rates (p < 0.05). Compared with DL-cloprostenol sodium, D-cloprostenol sodium shortened the farrowing duration and birth interval of sows for inducing farrowing (p < 0.05). Moreover, we observed that a single administration of both D-cloprostenol sodium and DL-cloprostenol sodium a day before delivery significantly reduced the rates of stillborn piglets type II in MS (p < 0.05). Compared to no treatment and single treatment with cloprostenol sodium, quartic treatments with cloprostenol sodium significantly increased the daily feed intake of MS, litter weight after weaning, and average daily gain of piglets (p < 0.05). Cloprostenol sodium improved the 21-day milk yield, with D-cloprostenol sodium showing the best effect, which increased lactation ability by 30.30% (176.72 kg vs. 135.63 kg) (p < 0.05). DL-cloprostenol sodium followed closely, increasing lactation ability by approximately 25.00% (169.71 kg vs. 135.63 kg) (p < 0.05). During lactation, sows administered with D-cloprostenol sodium observed increased serum prolactin levels. Compared to untreated sows, the sows administered with D-cloprostenol sodium and multiple DL-cloprostenol sodium visibly shortened the weaning-to-estrus interval (WEI) and weaning-to-service interval (WSI) (p < 0.05). Furthermore, quartic injections of D-cloprostenol sodium resulted in an 18 percentage point increase in the pregnancy rate of breeding sows compared to controls (82.61% vs. 64.58%) (p > 0.05). In summary, cloprostenol sodium could enhance the reproductive performance of MS, particularly in terms of lactation performance. Additionally, the effect of quartic injections of D-cloprostenol sodium was the most pronounced.

Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor.

Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible "contamination" of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.

Involvement of Porcine ß-Defensin 129 in Sperm Capacitation and Rescue of Poor Sperm in Genital Tract Infection.

In mammals, ß-defensins have been reported to play pivotal roles in sperm protection and fertilization. However, the function and mechanism of porcine ß-defensin 129 (pBD129) in the sperm remain unclear. Here, we demonstrate that pBD129 is a glycosylated protein and broadly exists in accessory sex glands and coats the sperm surface. We inhibited the pBD129 protein on the sperm surface with an anti-pBD129 antibody and found that sperm motility was not significantly affected; however, sperm acrosome integrity and tyrosine phosphorylation levels increased significantly with time (p < 0.05) during capacitation. These changes were accompanied by an increase in sperm Ca2+ influx, resulting in a significantly reduced in vitro fertilization cleavage rate (p < 0.05). Further investigation revealed that treatment with recombinant pBD129 markedly restored the sperm motility in semen contaminated with Escherichia coli. The results suggest that pBD129 is not only associated with poor sperm motility after genital tract infection but can also protect the spermatozoa from premature capacitation, which may be beneficial for semen preservation.

Carboxypeptidase E protein regulates porcine sperm Ca2+ influx to affect capacitation and fertilization.

Mammalian spermatozoa acquire their fertilizing ability in the epididymis, which is important for sperm maturation and capacitation. Carboxypeptidase E (CPE) is a prohormone-processing enzyme and sorting receptor that functions intracellularly. Recently, CPE was identified to exist in the seminal plasma. However, little is known about the effects of CPE on reproductive function. This study focused on the effects of CPE on sperm function and fertilization. Herein, CPE was identified to be localized in the boar sperm, testis, epididymis, accessory gonad and seminal plasma, with high expression found in the bulbourethral glands and cauda epididymis. Furthermore, compared with high motility spermatozoa, a decrease in CPE abundance was observed in low motile spermatozoa by Western blot analysis. The use of specific antibody to inhibit the CPE in spermatozoa led to a decrease in sperm motility, followed by an expected decrease in acrosome exocytosis and tyrosine phosphorylation in the capacitation process. These changes were accompanied by a decrease in intracellular Ca2+ ([Ca2+]i) influx, which resulted in a significant decrease in the cleavage rate during in vitro fertilization (IVF). Based on these observations, we suggest that CPE might affect porcine sperm Ca2+ influx to participate in the regulation of sperm function during capacitation.

Tubulin TUBB4B Is Involved in Spermatogonia Proliferation and Cell Cycle Processes.

Tubb4b (tubulin ß-4b chain) is essential for cell growth and development as a microtubule network protein. Previous studies have shown that TUBB4B affects mouse pronucleus migration, but the gene function has yet to be elucidated. To study TUBB4B-related functions in mouse reproductive development, we designed a single sgRNA in chromosome 2 and generated a knockout spermatogonia cell line of the ß-tubulin isoform Tubb4b by the CRISPR/Cas9 system. Tubb4b-KO spermatogonia recognized abnormal lysosomal membranes and cell morphology defects. Compared to control mouse spermatogonia, the proliferation rate was significantly slower and cycling stagnated in the G1/0 population. Although spermatogonia lacking TUBB4B have abnormal divisions, they are not lethal. We detected the mRNA levels of the cell-regulating cyclins CyclinsD1, CyclinsE, Cdk2, Cdk4, P21, Skp2 and the cell growth factors C/EBP α, C/EBP ß, and G-CSF in the spermatogonia of Tubb4b-KO and found that the expressions of CyclinsD1, Skp2 and cell growth factors were significantly reduced. Further analysis revealed that 675 genes were expressed differently after Tubb4b deletion and were enriched in negative regulation of cell population proliferation (GO:0008285), negative regulation of cell cycle G2/M phase transition (GO:1902750), and positive regulation of cell death (GO: 0010942). We also found that there is a common gene Cdkn1a (P21) in these three GO pathways related to cell proliferation and cell cycle, and both quantitative analysis and transcriptome sequencing results showed that the expression of this gene was up-regulated in Tubb4b knockout cells. This implies that Tubb4b may be involved in the division of spermatogonia with multiple cell cycle regulatory proteins. Overall, these data indicate that Tubb4b has a specific role in regulating spermatogonia proliferation and cell cycle.

Glutathione S-transferase kappa 1 is positively related with sperm quality of porcine sperm.

The glutathione S-transferase (GST) superfamily members play an important role in the male reproductive tract and sperm physiology. However, the expression profiles of some members of this protein family and their effect on sperm quality remain unclear. In this study, we found that GST kappa 1 (GSTK1) encoded protein is abundant in the testes and capacitated sperm acrosome. Western blot analysis revealed that the decreased abundance of GSTK1 was observed in low motile spermatozoa; moreover, GSTK1 expression decreased in sperm stored at 17°C under a long preservation time. In vitro analyses revealed that GSTK1 had no significant effect on sperm motility, capacitation, or acrosome reaction. Notably, after capacitated sperm were incubated with 4 and 8 µg/ml anti-GSTK1 antibodies, the fertilization rate significantly decreased in vitro fertilization assay. The current study demonstrates that GSTK1 is correlated with sperm quality and is a promising marker for the assessment of sperm quality and provides a basis for understanding the potential molecular mechanism for targeting pathogenic factors in male infertility.

Serum 25OHD3 of Obese Mice Is Affected by Liver Injury and Correlates with Testosterone Levels and Sperm Motility.

INTRODUCTION: The concentration of 25-hydroxycholecalciferol (25OHD3) in the serum of obese people is low and often accompanied by symptoms of low fertility. Therefore, vitamin D is recommended as a potential treatment option. However, after clinical trials, it was found that vitamin D cannot effectively increase the concentration of 25OHD3 in the serum of obese people. How obesity causes low 25OHD3 concentration and low fertility is unclear. METHODS: We analyzed the physiological and pathological changes in obese mice induced by a high-fat diet (HFD) and the changes in mice after supplementing with 25OHD3. RESULTS: The concentration of 25OHD3 in the serum of obese mice induced by HFD was significantly reduced, and these mice showed liver hypertrophy accompanied by abnormal liver injury, testicular hypertrophy, low testosterone levels, high leptin levels, and low sperm motility. The mRNA and protein expression of CYP2R1 of hydroxylated vitamin D3 was significantly reduced; CYP11A1 and CYP11A2, which synthesize testosterone, were significantly reduced. After supplementing with 25OHD3, there was an increase in serum 25OHD3 concentration, testosterone level, and sperm motility, but it cannot improve the degree of obesity, CYP2R1 expression, and liver damage. CONCLUSION: Our research shows that there is a metabolic interference mediated by 25OHD3 and testosterone between obesity and low sperm motility. The results of this study can provide a scientific basis for studying the mechanism of 25OHD3 and hormone regulation and treating obese people with low sperm motility.

A novel identified circ-ANKHD1 targets the miR-27a-3p/SFRP1 signaling pathway and modulates the apoptosis of granulosa cells.

The specific expression profile and function of circular RNAs (circRNAs) in mammalian ovarian follicles, especially during the atresia process, are unclear. In this study, we verified and explored the expression and function of circ-ANKHD1 in granulosa cells. Our results showed that abundance of circ-ANKHD1 was significantly lower in the granulosa cells than that of ANKHD1. The expression of ANKHD1 was highest in the granulosa cells from follicles with a diameter of 5-6 mm and lowest in that with a diameter of 3-4 mm. Furthermore, the expression level of circ-ANKHD1 in the ovarian tissue of 1-day-old piglets was significantly higher than that of 17-month-old multiparous sows. The luciferase reporter assay showed the potential interaction between circ-ANKHD1 and miR-27a-3p/miR-142-5p. Furthermore, circ-ANKHD1 overexpression up-regulated SFRP1 expression, while miR-27a-3p overexpression suppressed SFRP1 expression in granulosa cells. Circ-ANKHD1 overexpression significantly decreased the cell apoptotic rates of the granulosa cells and repressed the cell population at G0/G1 and S phases but increased cell population at G2/M phase. Finally, circ-ANKHD1 overexpression increased the mRNA expression levels of Bcl-2 and cyclin D1 in the granulosa cells, while there are no effects on the mRNA expression levels of caspase-3, p53, Bax, and proliferating cell nuclear antigen. In conclusion, our study for the first time identified a novel circRNA, circ-ANKHD1 that may be associated with the biological functions of granulosa cells. Circ-ANKHD1 may promote the granulosa cell proliferation, but attenuate apoptosis, and these effects may be associated with modulation of miR-27a-3p/SFRP1.

Investigation Into the Relationship Between Sperm Cysteine-Rich Secretory Protein 2 (CRISP2) and Sperm Fertilizing Ability and Fertility of Boars.

The proteins in the seminal plasma and on the sperm surface play important roles in sperm function and numerous reproductive processes. The cysteine-rich secretory proteins (CRISPs) are enriched biasedly in the male reproductive tract of mammals, and CRISP2 is the sole member of CRISPs produced during spermatogenesis; whereas the role of CRISP2 in fertilization and its association with fertility of boars are still unclear. This study aimed to investigate the relationship between the sperm CRISP2 and boar fertility, and explore its impact sperm fertilizing ability. The levels of CRISP2 protein in sperm were quantified by ELISA; correlation analysis was performed to evaluate the association between CRISP2 protein levels and boar reproductive parameters. Meanwhile, the expression of CRISP2 in boar reproductive organs and sperm, and the effects of CRISP2 on in vitro fertilization (IVF) were examined. The results showed that boars with high sperm levels of CRISP2 had high fertility. The protein levels of CRISP2 in sperm were positively correlated with the litter size (r = 0.412, p = 0.026), the number of live-born piglets (r = 0.421, p = 0.023) and the qualified piglets per litter (r = 0.381, p = 0.042). CRISP2 is specifically expressed in the testis and sperm of adult boars, and its location on sperm changed mainly from the post-acrosomal region to the apical segment of acrosome during capacitation. The cleavage rate was significantly decreased by adding the anti-CRISP2 antibody to the IVF medium, which indicates CRISP2 plays a critical role in fertilization. In conclusion, CRISP2 protein is specifically expressed in the adult testis and sperm and is associated with sperm fertilizing ability and boar fertility. Further mechanistic studies are warranted, in order to fully decipher the role of CRISP2 in the boar reproduction.

Perfluorooctanoic acid inhibits the maturation rate of mouse oocytes cultured in vitro by triggering mitochondrial and DNA damage.

BACKGROUND: Perfluorooctanoic acid (PFOA) is widely used in the manufacture of household and industrial products. It has certain toxicity and leaves many residues in the environment. Numerous studies have shown that PFOA exhibits endocrine disrupting properties and immunotoxicity and induces developmental defects. However, there is very little information regarding its toxicity on oocytes. METHODS: We cultured denuded oocytes in maturation medium supplemented with 0, 300, or 500 PFOA during IVM and evaluated the maturation of oocytes from the aspects of ROS(DCFH-DA), mitochondria(MitoOrange and JC-1), DNA damage(P-H2AX), and cytoskeleton(ß-tubulin). RESULTS: Compared with the control group, the PFOA treatment group exhibited significantly reduced proportion of oocytes matutation. Furthermore, the DCFH-DA test showed that PFOA significantly increased reactive oxygen species (ROS) levels. PFOA disrupted mitochondrial distribution and decreased mitochondrial function as assessed using MitoOrange and JC-1. In addition, PFOA-treated oocytes exhibited a significantly higher percentage of P-H2AX, defective ß-tubulin, abnormal chromosome alignment, lower expression of the anti-apoptotic gene Bcl-2, and higher expression of the apoptotic genes caspase3 and Bax. CONCLUSION: In summary, PFOA could negatively and directly affect oocyte maturation in vitro and cause oxidative stress, mitochondrial function disruption, DNA damage, cytoskeleton damage, and apoptosis.

Epigallocatechin-3-Gallate Promotes the in vitro Maturation and Embryo Development Following IVF of Porcine Oocytes.

PURPOSE: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and exerts protective effects because of its strong antioxidant properties. As far as we know, there is still a lack of systematic research on the effects of EGCG on the in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes. The present study aimed to determine the effects of EGCG on the IVM and IVF of porcine oocytes. METHODS: Porcine oocytes were treated with different concentrations of EGCG (5, 10 and 20 µM), and the cumulus cell expansion, oocyte maturation rate, reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) levels, total antioxidant capacity were determined. The mRNA expression levels of oxidative stress- and apoptosis-associated genes were determined by quantitative real-time PCR. The cleavage rate and blastocyst rate of oocytes after 10 µM EGCG treatment during IVM and IVF were also evaluated. RESULTS: EGCG at 5, 10 and 20 µM significantly promoted cumulus cell expansion, and EGCG at 10 µM increased the oocyte maturation rate. EGCG (10 µM) treatment reduced the ROS and MDA levels, while increased the antioxidant capacity and GSH concentrations in the mature oocytes. The qRT-PCR results showed that EGCG treatment up-regulated the mRNA expression of catalase, glutathione peroxidase and superoxide dismutase in the mature oocytes. In addition, EGCG treatment also decreased the mRNA expression levels of Bax and caspase-3 and increased the Bcl-2 mRNA expression level in the mature oocytes. In addition, the cleavage rate and blastocyst rate of oocytes treated with 10 µM EGCG during IVM and IVF were significantly higher than those of the control group. CONCLUSION: Our results suggest that EGCG promotes the in vitro maturation and embryo development following IVF of porcine oocytes. The protective effects of EGCG on the oocytes may be associated with its antioxidant and anti-apoptosis properties.

Analysis of differentially abundant proteins related to boar fertility in seminal plasma using iTRAQ-based quantitative proteomics.

Animal fertility is one of the most important characteristics for the livestock breeding industry. Conventional semen analysis provides basic information on sperm quality, but the predictive value of such analysis with regard to fertility remains questionable. Therefore, it is important to determine and predict male fertility more accurately in the clinic. To identify seminal plasma proteins involved in fertility, isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography with tandem mass spectrometry (quantitative proteomic analysis) were used to identify differentially abundant proteins (DAPs) in seminal plasma between high- and low-reproductive-efficiency Landrace boars. A total of 141 DAPs were identified, of which 125 upregulated and 16 downregulated proteins were subjected to bioinformatics analysis. These DAPs were found to be mainly involved in proteolysis, ATP binding, and energy metabolism. We investigated the relevance of three DAPs-ceruloplasmin, carboxypeptidase E (CPE), and serpin family A member 12 (SERPINA12)-in an in vitro fertility assay. This assay revealed that the inhibition of these proteins with antibodies can reduce or increase the fertilization rate. These results indicate possible biomarkers for the selection of high-fertility boars and provide a theoretical basis for the use of protein biomarkers in the livestock breeding industry. SIGNIFICANCE: Our study identified differentially abundant proteins in the seminal plasma of high-reproductive-efficiency and low-reproductive-efficiency Landrace boars. These proteins may be used as biomarkers to screen out high-fertility boars. The study can provide not only a new method for improving the effects of artificial insemination and reproductive efficiency of boars but also an important reference for boar breeding. Meanwhile, because pigs and humans have similar physiological parameters and organ sizes, our findings can also serve as a reference for human reproduction research.

GPx6 is involved in the in vitro induced capacitation and acrosome reaction in porcine sperm.

Glutathione peroxidases (GPxs) are regarded as important protectors against oxidative stress. Some members of this protein family were reported to play key roles in protecting sperm against oxidative stress. Whether GPx6 a member of the GPx family also plays a role in protection against oxidative stress is not known to date. The objective of the present study was to evaluate the localization and function of glutathione peroxidase 6 (GPx6) in boar accessory sex glands, seminal plasma, and sperm, as well as the effect of GPx6 on vitality and capacitation in boar sperm. qPCR and Western blot analysis demonstrated the presence of GPx6 in testis, epididymis, bulbourethral glands, prostate, seminal vesicle, sperm and seminal plasma. Incubation of sperm with an GPx6 antibody had no significant effect on the viability of boar sperm prior to capacitation. Surprisingly, when capacitated sperm was incubated with the GPx6 antibody for 240 min, sperm vitality was significantly improved. Western blotting showed that in capacitated sperm without prior pretreatment, GPx6 protein content was reduced compared to sperm before capacitation. To further confirm a role for GPx6 in sperm capacitation, we tested sperm acrosome reaction by ACR.2 and FITC-PSA. The results showed that treatment of sperm with the GPx6 antibody significantly increased sperm capacitation and acrosome reaction. Furthermore, we examined the concentration of cAMP in sperm after capacitation. ELISA demonstrated that the cAMP concentration in the sperm exposed to the GPx6 antibody was significantly higher than that of the control group. In addition, the exposure of sperm to the GPx6 antibody significantly increased the concentration of H2O2, while the expression of SOD3 and CAT were decreased. Based on these observations we would like to postulate that in the boar reproductive tract the GPx6 protein becomes attached to the sperm head preventing the sperm to undergo premature capacitation by affecting components of the antioxidant pathway. How GPx6 expression following ejaculation becomes suppressed to allow sperm capacitation to take place needs further investigation.

Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions-circ_CBFA2T2 and circ_KIF16B-were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes-the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Effects of 25-hydroxycholecalciferol supplementation in maternal diets on reproductive performance and the expression of genes that regulate lactation in sows.

One hundred Yorkshire × Landrace sows were randomly assigned to one of two dietary treatments (diet ND: 6,000 IU vitamin D3 /d feed; diet 25-D: 200 µg/day 25OHD3 feed). The experiment began on d 90 of gestation and continued until weaning on day 21 of lactation. In sows that received 25OHD3 , the growth rate of the piglets before weaning was significantly accelerated (0.266 kg/day, p < .05). Sow serum was collected after weaning, and those in the 25OHD3 group were found to have significantly higher serum calcium (CA) and phosphorus (PI) levels (p < .05). Interestingly, the oestrus cycle of sows fed 25OHD3 was significantly shortened (p < .05), the oestrus time was concentrated on the fifth day after weaning, and the piglets were born with a higher degree of uniformity (p < .05). Colostrum was collected on the day of delivery, and the colostrum of sows fed 25OHD3 contained higher milk fat content than the control group (p < .05). 25OHD3 supplementation increased the mRNA and protein expression of INSIG1 and SREBP1, which regulate milk fat synthesis, in the mammary gland of lactating sows (p < .05). In conclusion, 25OHD3 supplementation in maternal diets improved reproductive performance, milk fat content and the mRNA and protein levels of genes regulating milk fat synthesis in lactating sows.

Identification of new protein biomarkers associated with the boar fertility using iTRAQ-based quantitative proteomic analysis.

In this study, we performed the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis in the spermatozoa of Landrace boars with different fertility potentials and investigated the ability of sperm acrosome associated 4 (SPACA4) and IZUMO family member 2 (IZUMO2) to predict the reproductive perform of boars. The iTRAQ results revealed that 202 proteins were up-regulated and 43 proteins were down-regulated in the spermatozoa from high fertility boars. SPACA4 and IZUMO2 protein levels were significantly up-regulated in the spermatozoa from high fertility boars. SPACA4 and IZUMO2 expression were specifically detected in the adult boar testis. SPACA4 levels were positively correlated with Sow's farrowing rate and reproductive efficiency, but not litter size. IZUMO2 were positively correlated with litter size, Sow's farrowing rate and reproductive efficiency. Treating the boar semen with SPACA4 or IZUMO2 antibodies for 30 min and 60 min failed to affect the sperm motility; while treating the semen with SPACA4 antibody significantly reduced the fertilization and cleavage rates. Similar results for fertilization and cleavage rates were found in IZUMO2 antibody-treated semen. Collectively, our results indicated that protein levels of SPACA4 and IZUMO2 in the spermatozoa were positively related to the reproductive performance of Landrace boars.

Differential expression profiles of long non­coding RNAs during the mouse pronuclear stage under normal gravity and simulated microgravity.

Pronuclear migration, which is the initial stage of embryonic development and the marker of zygote formation, is a crucial process during mammalian preimplantation embryonic development. Recent studies have revealed that long non­coding RNAs (lncRNAs) serve an important role in early embryonic development. However, the functional regulation of lncRNAs in this process has yet to be elucidated, largely due to the difficulty of assessing gene expression alterations during the very short time in which pronuclear migration occurs. It has previously been reported that migration of the pronucleus of a zygote can be obstructed by simulated microgravity. To investigate pronuclear migration in mice, a rotary cell culture system was employed, which generates simulated microgravity, in order to interfere with murine pronuclear migration. Subsequently, lncRNA sequencing was performed to investigate the mechanism underlying this process. In the present study, a comprehensive analysis of lncRNA profile during the mouse pronuclear stage was conducted, in which 3,307 lncRNAs were identified based on single­cell RNA sequencing data. Furthermore, 52 lncRNAs were identified that were significantly differentially expressed. Subsequently, 10 lncRNAs were selected for validation by reverse transcription­quantitative polymerase chain reaction, in which the same relative expression pattern was observed. The results revealed that 12 lncRNAs (lnc006745, lnc007956, lnc013100, lnc013782, lnc017097, lnc019869, lnc025838, lnc027046, lnc005454, lnc007956, lnc019410 and lnc019607), with tubulin ß 4B class IVb or actinin α 4 as target genes, may be associated with the expression of microtubule and microfilament proteins. Binding association was confirmed using a dual­luciferase reporter assay. Finally, Gene Ontology analysis revealed that the target genes of the differentially expressed lncRNAs participated in cellular processes associated with protein transport, binding, catalytic activity, membrane­bounded organelle, protein complex and the cortical cytoskeleton. These findings suggested that these lncRNAs may be associated with migration of the mouse pronucleus.

Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells.

BACKGROUND: Male germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells. METHODS: Porcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis. RESULTS: We identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs). CONCLUSION: Collectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.

Serial Culture Is Critical for In Vitro Development of Parthenogenetic Embryos in the Golden Syrian Hamster.

Unlike oocytes of many other mammalian species, parthenogenetically activated hamster oocytes have not been reported to develop beyond the two-cell stage. This study investigated the in vitro development into blastocysts of parthenogenetic embryos of Golden Syrian hamsters. We observed that hamster oocytes could easily be artificially activated (AA) by treatment with ionomycin plus 6-dimethylaminopurine + cycloheximide + cytochalasin B as assessed by embryo cleavage in HECM-9 (63.15%) or HECM-10 (63.82%). None of the cleaved embryos developed beyond the two-cell stage when cultured in either of the two media. However, some of the embryos overcame the two-cell block and developed to the blastocyst stage (26.45%) when they were first cultured in HECM-10 for 24 hours and then in HECM-9 (serial culture media HECM-10-9) for 72 hours. Blastocyst development was further significantly (66.2%) improved when embryos were cultured in HECM-10 supplemented with ethylenediaminetetraacetic acid for 24 hours, then in HECM-9 supplemented with glucose for 72 hours (serial culture media HECM-11a-b). Hamster oocytes activated with ionomycin, ethanol, or a combination of the two treatments would develop to the blastocyst stage in serial culture media HECM-11a-b, whereas none of the spontaneously activated oocytes cleaved (0% vs. 86.93%, p < 0.05). DNA and microtubule configurations of spontaneously activated and AA oocytes were assessed by immunocytochemical staining and fluorescence microscopy. The results indicate that serial culture and the method of activation are critical for overcoming the in vitro developmental block of hamster parthenogenetic embryos. This study is the first to report blastocyst development from parthenogenetically activated hamster oocytes.