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.

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.

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.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis.

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = -6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

[Heat shock protein 90 in male reproduction].

Heat shock protein 90 (Hsp90), as a member of the Hsp family and widely found in the gonads of humans and animals, plays an essential role in male reproduction and induces various changes in the process of reproduction. Hsp90 regulates the division of germ cells and participates in spermatogenesis, location of germ cells, formation of sperm microtubes, protection of sperm from oxidative stress, and inducement of acrosomal reaction. Studies showed significant changes in location and expression of Hsp90 in the sperm of oligospermia and asthenospermia patients. This paper presents an overview of Hsp90 in male reproduction and male infertility and a prospect of the treatment of male infertility.

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 102 copies/µL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.

Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

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.

[Role of CYCS in the cytogenesis and apoptosis of male germ cells and its clinical application].

In order to maintain the stability of intracellular environment, various cells die autonomously in vivo, which is called apoptosis. As a key factor of cell apoptosis in the mitochondrial pathway, cytochrome c, somatic (CYCS) plays an important role in the process of male spermatogenesis. Based on more than 40 recent studies on CYCS by Chinese and foreign scholars, this review focuses on the involvement of CYCS in the mechanisms of the apoptosis and anti-apoptosis of germ cells and in the regulation of their quality and activity and also outlines the role of CYCS in marking male reproductive performance as well as in male infertility and its underlying mechanisms.

Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus.

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/µL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Synthesis, Photothermal Effect and Cytotoxicity of Fe3O4@Au Nanocomposites.

It is currently a very active research area to develop multifunctional nanocomposites (NCs) which integrate the novel properties from various nanomaterials for multimodal imaging and simultaneous therapy. These theranostic nanoplatforms can provide complementary information from each imaging modality for accurate diagnosis and, at the same time, afford an imaging-guided focused tumor therapy. Among them, core/shell Fe3O4@Au NCs have attracted wide attention due to their unique advantages in magnetic targeting, multimodal imaging and photothermal therapy. This study developed a layer-by-layer assembling approach to synthesize Fe3O4@Au NCs with high photothermal conversion efficiency. The as-synthesized NCs showed significant photothermal ablation capability to HeLa cells in vitro under near infrared laser irradiation. To ensure the safety for medical applications, the bio-effects of Fe3O4@Au NCs on RAW264.7 cells were carefully assessed, in terms of cell viability, oxidative stress and apoptosis. We have demonstrated that Fe3O4@Au NCs had good biocompatibility in RAW264.7 cells and no significant cytotoxicity was found. Therefore, the Fe3O4@Au NCs synthesized in this study have great potential as an ideal candidate for CT/MR imaging and photothermal therapy.

Development and imprinted gene expression in uniparental preimplantation mouse embryos in vitro.

Increasing numbers of reports show that imprinted genes play a crucial role in fetal development, and uniparental embryos, which possess two paternally or two maternally derived pronuclei, are excellent tools for investigating the biological significance of imprinted genes. In the present study, to examine the in vitro developmental ability and expression pattern of eight imprinted genes in uniparental embryos, we produced androgenones, gynogenones, and parthenogenones using enucleation. Our data confirmed the previously observed restriction in haploid androgenetic development potential and first indicated that diploid androgenetic embryos were arrested in the 3/4-cell stage. Some imprinted genes were expressed in androgenetic, gynogenetic, and parthenogenetic blastocysts, suggesting that they were unable to maintain their imprinted expression status in uniparental embryos and that both paternal and maternal alleles are required for the specific expression of some imprinted genes.

[The progress of sperm functional proteins regulating the process of fertilization].

The process of mammalian fertilization involves a series of sperm functional activities, including the sperm transportation, hyperactivation and capacitation, acrosome reaction and sperm-egg fusion, etc. The semen proteins play indispensable roles in these processes, and they are closely associated with the fecundity of males. So these proteins can be biomarkers to evaluate the fertilization capacity of mammalian semen. In this review, we mainly introduce some semen proteins relevant to spermatozoa functions and illustrate their important regulatory roles on the fertilization processes, involving spermatozoa motility, capacitation, acrosome reaction, penetrating the zona pellucida and sperm-egg fusion, etc. Moreover, their potential applications in the evaluation of heredity in livestock are also summarized.

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.

Tert-Butylhydroquinone Mitigates T-2-Toxin-Induced Testicular Dysfunction by Targeting Oxidative Stress, Inflammation, and Apoptosis in Rats.

Tert-butylhydroquinone (tBHQ) has emerged as a promising candidate for mitigating the adverse effects of T-2-induced reproductive toxicity. The protective effects of tBHQ on rat sperm quality, testicular injury, apoptosis, and inflammation induced by T-2 toxin exposure were investigated. Histopathological examination of testicular tissues revealed severe damage in the T-2-treated group, characterized by disorganized germ cell arrangement, thinning of the convoluted seminiferous tubule walls, and significant cellular necrosis. However, tBHQ administration, either as a preventive or therapeutic measure, mitigated this structural damage. Image analysis confirmed an increase in the cross-sectional area and height of the convoluted seminiferous tubules in the tBHQ-treated groups compared to the T-2-treated group (p < 0.05), indicating tBHQ's efficacy in alleviating testicular damage. Additionally, tBHQ treatment significantly inhibited T-2-induced apoptosis of testicular tissue cells, as evidenced by the results showing reduced apoptotic cell counts and downregulation of the BAX/BCL2 ratio and caspase-3 expression (p < 0.05). tBHQ significantly increased the concentrations of the antioxidant factors SOD, CAT, TAC, and GSH-PX. Furthermore, tBHQ attenuated the inflammatory response induced by T-2 exposure, as indicated by the decreased mRNA expression of the proinflammatory cytokines Tnf, Il1, and Il10 in testicular tissue (p < 0.05). Additionally, tBHQ treatment alleviated the decline in serum testosterone induced by the T-2 and promoted testosterone synthesis gene expression, including for the genes 17ß-HSD and Cyp11a1, in rat testes (p < 0.05). These findings underscore tBHQ's role as a therapeutic agent combatting T-2-induced reproductive toxicity, highlighting its antioxidative, anti-apoptotic, and anti-inflammatory properties. Further elucidation of tBHQ's mechanisms of action may offer novel strategies for preventing and treating reproductive disorders induced by environmental toxins.

[The effect of microRNAs on the regulatory network of pluripotency in embryonic stem cells].

Embryonic stem cells (ESCs) are pluripotent stem cells characterized by their ability to self-renew and their pluripotency to differentiate into all cell types. MicroRNA (miRNA) is a small non-coding RNA molecule which can regulate transcriptional and post-transcriptional gene expression, and may also play significant roles in regulating proliferation and differentiation of ESCs. The maintenance of pluripotency in ESCs may involve a regulatory network of many factors and pathways regulated by miRNA, which includes ESCs transcription factors, cell cycle regulation, epigenetic modifications as well as intracelluar signal transduction. This review mainly elaborates the biogenesis of miRNA, the miRNA families regulating the pluripotency of ESCs, and the effect of miRNA on the regulatory network of pluripotency in ESCs.

Quantifying the contribution of ecological water replenishment on aquifer recovery using a refined groundwater model.

Due to its independent control and directly easy operation, ecological water replenishment (EWR) has been an important measure for restoring river ecosystems. However, the positive and negative contribution of the EWR activities to aquifer system are not fully understood under the combined influences of climate change and human activities across time scales. A refined groundwater flow model integrating an open channel flow at daily time scales is developed in a part of Northern China Plain to reproduce the dynamic process of groundwater level changes. After model calibration with groundwater level and runoff data, the changes of simulated groundwater level and river runoff have the Nash-Sutcliffe efficiency coefficient of 0.98 and 0.60, respectively. Results clearly demonstrate that the impulse response of aquifer recovery to runoff in three centralized EWRs. By using with and without EWR method, the simulated maximum contribution of EWR near river to aquifer recovery may be over 70 %. Scenario analysis method considering different precipitation, groundwater exploitation reduction and EWR activities are applied to evaluate the total quantities of aquifer recovery. The prediction of nine-year EWR activities under multiple scenarios shows that the increased groundwater level generally varies from 4.08 to 8.57 m, and the contribution of EWR accounts for 7.88 %-36.59 %. It is also noticed that 14 out of the 18 informal landfill sites will face potential groundwater pollution risks, indicating the negative influences of long-term EWR activities. This study can provide a method for quantifying the influences and contribution of EWR on aquifer recovery and can be referred to as a guideline for EWR evaluation with similar hydrogeological conditions.

[Tail-suspended model simulating mouse oocytes maturation inhibited with microgravity].

We studied the effects of simulated microgravity on mouse oocytes maturation, and analyzed whether the tail-suspended model can be applied to investigate simulated microgravity effects on reproductive processes in female mice. Mouse oocytes were cultured in vitro with microgravity simulated by a rotating wall vessel bioreactor and by tail-suspended model, and the maturation rate of the mouse oocytes in the two models were examined in vivo. The maturation rate of mouse oocytes cultured in simulated microgravity was 8.93%, and that was 72.33% in 1g gravity. In ratio, oocyte maturation rate had no significant difference between the rotational group and control group. Microgravity simulated by the tail-suspended model inhibited mouse oocytes maturation and increased the rate of oocytes abnormity. The maturation rate of tail-suspended mouse oocytes was 14.54%, which was significantly lower than that of control group. Tail-suspended model should be an ideal model to investigate simulated microgravity effects on reproductive processes of female mice.

Osteopontin enhances sperm capacitation and in vitro fertilization efficiency in boars.

In this study, we used more reliable experimental materials and methods to detect the effects of osteopontin (OPN) on boar sperm in vitro capacitation, acrosome reaction, and fertilization efficiency. We reorganized and obtained the OPN protein of the porcine source. Immunofluorescence and Western blot show the localization and expression of the OPN protein before and after sperm capacitation. To determine whether OPN can affect sperm during sperm capacitation, we examined cyclic adenosine monophosphate (cAMP) concentrations after sperm capacitation, and the results showed that OPN significantly increased the cAMP concentration in sperm (p < 0.05). Flow cytometry showed that 0.1 µg/mL OPN-treated sperm had better acrosome reaction ability. In vitro fertilization (IVF) showed that 0.1 µg/mL OPN significantly increased the rate of embryo division. In conclusion, this study found that 0.1 µg/mL porcine OPN protein can significantly improve porcine capacitated sperm motility, cAMP concentration after capacitation sperm, acrosome reaction ability, and embryo division during IVF and provides new clues to explore the mechanism of OPN's function on sperm.

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.