Mating Tests Research Articles

Transcriptomics of tissue exosomes to investigate miR-195-5p’s amelioration of endometrial fibrosis via the YAP-Smad7 pathway: an animal study

BackgroundA significant research gap exists regarding the role of tissue exosomes in intrauterine adhesions (IUAs). This study aims to investigate the involvement of miR-195-5p and its regulatory network in IUAs through the analysis of tissue exosomes.MethodsExosomes from rat uterine tissue with intrauterine adhesions were analyzed via transcriptomics to identify downstream target genes of miR-195-5p, cross-referencing with the human endometrial transcriptomics database GSE224093. Dual luciferase labeling confirmed miRNA-target gene interactions. The therapeutic efficacy of a miR-195-5p agonist was assessed in vivo through HE staining, Masson staining, and mating tests. The mechanisms underlying extracellular matrix (ECM) deposition and myofibroblast transdifferentiation in endometrial fibrosis were investigated both in vitro and in vivo using RT-PCR, Western Blot, immunofluorescence, and immunohistochemistry. Migration ability of endometrial stromal cells was evaluated using CCK8, scratch tests, and Transwell assays. Finally, the clinical potential of miR-195-5p was compared with autologous adipose-derived mesenchymal stem cells.ResultsThe expression of miR-195-5p in uterine tissue exosomes from intrauterine adhesions was found to be decreased. Treatment with a miR-195-5p agonist resulted in improved endometrial health, reduced fibrosis, increased glandular density, and enhanced birth rates in rats. Both in vivo and in vitro experiments confirmed that miR-195-5p decreased ECM deposition, reduced myofibroblast transdifferentiation, and inhibited the migration of endometrial stromal cells. This was achieved through the downregulation of YAP expression in the Hippo pathway and the upregulation of Smad7. Notably, the therapeutic efficacy of miR-195-5p agonists was comparable to that of stem cell therapy, offering promising avenues for clinical application.ConclusionsDifferential expression of miR-195-5p in tissue exosomes can reduce ECM deposition and myofibroblast transdifferentiation, improving endometrial fibrosis by regulating the YAP-Smad7 pathway in the Hippo signaling cascade.

Phylogeny and species diversity of Armillaria in China based on morphological, mating test, and GCPSR criteria

ABSTRACT More than 600 Chinese specimens of Armillaria were identified by mating tests, Genealogical Concordance Phylogenetic Species Recognition (GCPSR), and comparison of morphological characteristics. Sixteen Chinese Biological Species (CBS) of Armillaria were identified by 30,340 mate pair combinations. Fifteen Chinese Phylogenetic Species (CPS) were recognised based on Independent Evolutionary Lineage (IEL) recognition and concatenated six-gene analysis (actin, h3h, hisps, LSU rDNA, rpb1, and tef1α). All the biological species and phylogenetic species were identical and possessed the same species boundary, except for CBS K (A. mellea) and CBS G (A. mellea ssp. nipponica) which were the same phylogenetic species. On the basis of CBS and CPS, eight new species of Armillaria in China were distinguished using macro and micro morphology, and they are described as A. algida, A. amygdalispora, A. bruneocystidia, A. luteopileata, A. pungentisquamosa, A. sinensis, A. tibetica, and A. violacea. This study indicates that the GCPSR approach provides the same resolution as mating tests in identification of Armillaria species.

Eighteen genes primarily expressed in the testis are not required for male fertility in mice†.

There are approximately 20000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

Armillaria korhonenii, the sixteenth biological species of Armillaria from China

Armillaria korhonenii, designated as Chinese biological species Q (CBSQ) here, is described as a new species based on phylogenetic and morphological evidence as well as the results of mating tests. This species is the sixteenth biological species of Armillaria reported from China following the discovery of 15 previously known biological species. Phylogenetically, A. korhonenii is the earliest divergent lineage of the genus Armillaria as inferred from the nucleotide sequences of the Elongation Factor 1-Alpha (EF1a). Morphologically, it is characterized by its ellipsoid to elongate basidiospores, yellowish orange to honey yellow pileus with fibrillose squamules. Mating tests indicated that A. korhonenii is a tetrapolar heterogeneous species and is inter-sterile with all the tested species except partial compatibility with CBSH.

PABPN1L is required for maternal mRNA degradation after meiosis resumption.

Poly(A)-binding proteins (PABPs) play roles in mRNA maturation, translational activity, and decay. The functions of PABPs, especially PABPN1 and PABPC1, in somatic cells have been well-studied. However, little is known about the roles of PABPs in oocytes because of the unique mechanisms of mRNA metabolism in oocytes. This study focused on PABPN1L and generated Pabpn1l knockout (KO) mice using the CRISPR/Cas9 system. After mating tests, we found that Pabpn1l KO females were infertile due to the failure of the embryos to develop to the 4-cell stage. RNA-seq analysis revealed aberrant mRNA persistence in Pabpn1l KO-MII oocytes, which indicates impaired mRNA degradation during the germinal vesicle (GV) to MII transition. We also revealed that the exogenous expression of Pabpn1l mRNA in KO-GV oocytes recovered defects of embryonic development. PABPN1L is partly indispensable for female fertility in mice, owing to its necessity for embryonic development, which is supported by mRNA degradation during GV to MII maturation.

Disruption of testis-enriched cytochrome c oxidase subunit COX6B2 but not COX8C leads to subfertility.

Mammalian sperm flagellum contains the midpiece characterized by a mitochondrial sheath that packs tightly around the axoneme and outer dense fibers. Mitochondria are known as the "powerhouse" of the cell, and produce ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). However, the contribution of the TCA cycle and OXPHOS to sperm motility and male fertility is less clear. Cytochrome c oxidase (COX) is an oligomeric complex localized within the mitochondrial inner membrane, and the terminal enzyme of the mitochondrial electron transport chain in eukaryotes. Both COX6B2 and COX8C are testis-enriched COX subunits whose functions in vivo are poorly studied. Here, we generated Cox6b2 and Cox8c knockout (KO) mice using the CRISPR/Cas9 system. We examined their fertility and sperm mitochondrial function to determine the significance of testis-enriched COX subunits in male fertility. The mating test revealed that disrupting COX6B2 induces male subfertility, while disrupting COX8C does not affect male fertility. Cox6b2 KO spermatozoa showed low sperm motility, but mitochondrial function was normal according to oxygen consumption rates. Therefore, low sperm motility seems to cause subfertility in Cox6b2 KO male mice. These results also indicate that testis-enriched COX, COX6B2 and COX8C, are not essential for OXPHOS in mouse spermatozoa.

PM 2.5 juvenile exposure–induced spermatogenesis dysfunction by triggering testes ferroptosis and antioxidative vitamins intervention in adult male rats

PM2.5 derived from automobile exhaust can cause reproductive impairment in adult males, but the toxic effects of PM2.5 exposure on reproductive function in juvenile male rats and its relationship with ferroptosis have not been reported. In this paper, 30-day-old juvenile male Sprague-Dawley (SD) rats were divided into four groups (blank control, vitamin control, PM2.5, and PM2.5+Vitamin). The blank control group was fed normally, and the vitamin control group was given intragastric administration of vitamins in addition to normal feeding. PM2.5 was administered via tracheal intubation. When the rats were treated for 4 weeks until reaching the period of sexual maturity. A mating test was performed first, and then their testicular and epididymal tissues were studied. Compared with control rats, juvenile male rats exposed to PM2.5 showed a decreased sperm count and fertility rate, redox imbalance, damaged mitochondria, a metabolic disorder of intracellular iron ions, and a significant rise in ferroptosis during the period of sexual maturity. After antioxidative vitamins intervention, the redox imbalance, metabolic disorder of intracellular iron ions, and ferroptosis were all alleviated, leading to the following conclusions: after being exposed to PM2.5 from automobile exhaust, male juvenile rats during the period of sexual maturity have significantly decreased reproductive function. The reproductive toxicity of PM2.5 is closely related to oxidative stress and ferroptosis. In addition, ferroptosis decreases and reproductive function is recovered to some degree after antioxidative vitamins intervention.Graphical abstract

Being friendly: paced mating for the study of physiological, behavioral, and neuroplastic changes induced by sexual behavior in females.

Paced mating in rats is an experimental condition that allows the evaluation of sexual behavior in a way that closely resembles what occurs in seminatural and natural conditions enabling the female to control the rate of the sexual interaction. In conventional non-paced mating tests, females cannot escape from male approaches, which may lead to an unrewarding overstimulation. Paced mating is an alternative laboratory procedure that improves animal welfare and has a higher ethological relevance. The use of this procedure contributed to the identification of physiological and behavioral factors that favor reproduction. Paced mating includes motivational and behavioral components differentiating quantitative and qualitative characteristics that are critical for the induction of the rewarding properties of mating. These positive consequences ensure that the behavior will be repeated, favoring the species' survival. Sexual reward is an immediate consequence of paced mating, mediated mainly by the endogenous opioid system. Paced mating also induces long-lasting neuroplastic changes, including gene expression, synthesis of proteins, and neurogenesis in sex-relevant brain areas. The interest in paced mating is growing since the complexity of its elements and consequences at different levels in a laboratory setting resembles what occurs in natural conditions. In this review, we analyze the classic studies and recent publications demonstrating the advantages of using paced mating to evaluate different aspects of sexual behavior in females.

Spermidine suppresses oxidative stress and ferroptosis by Nrf2/HO-1/GPX4 and Akt/FHC/ACSL4 pathway to alleviate ovarian damage

AimsOxidative stress is considered to be one of the culprits of ovarian dysfunction. Spermidine (SPD) is a natural aliphatic polyamine that is widely present in living organisms and has been shown to exert preventive effects on various ageing-related diseases. This study seeks to investigate the potential preventive and protective effects of SPD on ovarian oxidative damage. Main methodsOvarian oxidative stress model in C57BL/6 mice was established by 3-nitropropionic acid. Female mice were administrated 10 mg/kg or 15 mg/kg SPD. The estrous cycle, serum hormone levels and mating test were measured to evaluate ovarian function. Follicle counts and AMH levels to assess ovarian reserve. Masson's trichrome to assess ovarian fibrosis. TUNEL analysis to evaluate follicular granulosa cells (GCs) apoptosis. Oxidative stress and autophagy indicators (Nrf2, HO-1, GPX4, LC3B, P62) were measured in vivo and in vitro. RNA-sequencing was performed on SPD-treated GC to study the effects of SPD on Akt and FHC/ACSL4 signaling. Key findingsSPD supplementation improved ovarian endocrine function and reproductive capacity in oxidative stress mice. SPD regularized the estrous cycle and alleviated oxidative stress. Furthermore, SPD increased the ovarian reserve, reducing GC apoptosis by activating the Nrf2/HO-1/GPX4 pathway. RNA-sequencing showed that SPD induced 230 genes changes in porcine GC, which were mainly involved in oocyte meiosis, arginine biosynthesis and glutathione metabolism pathways. SPD attenuated H2O2-induced ferroptosis by regulating Akt/FHC/ACSL4 signaling. SignificanceSPD alleviates oxidative stress and ferroptosis by regulating the Nrf2/HO-1/GPX4 and Akt/FHC/ACSL4 pathway, which may be a novel potential strategy to protect ovarian oxidative damage.

Brown adipose tissue-derived exosomes delay fertility decline in aging mice.

Ovarian aging has steadily grown to be a significant health issue for women as a result of the increase in average life expectancy and the postponement of reproductive age. One of the important pathological foundations of ovarian aging is formed by mitochondrial dysfunction, which causes decreases in follicle quantity and oocyte quality. In recent years, brown adipose tissue (BAT) transplantation has been proven as an effective treatment for aging-related diseases, such as ovarian aging. However, BAT transplantation is an invasive operation with long-term risks. Therefore, we need to find an alternative strategy. We injected BAT-derived exosomes into eight-month-old C57BL/6 female mice. The fertility was detected by the estrous cycle and mating test. The changes of ovary and oocyte were measured by ovarian volume, organ coefficient, follicle counting, and oocyte maturation rate. ROS level, mitochondrial membrane potential and ATP level were measured to analyze the mitochondrial function of oocytes. The changes in metabolism were explored by cold stimulation test, body weight and blood sugar. The possible molecular mechanism was further investigated by RNA sequencing. In terms of fertility, the estrous cycle of aging mice after BAT-derived exosome intervention was more regular, and the number of progenies and litters was increased. At the tissue level, the ovaries in the BAT-exosome group were larger, and the number of primordial follicles, secondary follicles, antral follicles and total follicles increased. At the cellular level, BAT-derived exosomes improved the maturation of oocytes in vivo and in vitro, increased the mitochondrial membrane potential and ATP levels of oocytes, and decreased ROS levels. Besides, BAT-derived exosomes ameliorated the metabolism and viability of aging mice. Furthermore, mRNA sequencing showed that BAT exosomes altered the expression levels of genes related to metabolism and the quality of oocytes. BAT-derived exosomes enhanced mitochondrial function, promoted follicle survival, improved fertility, and extended ovarian lifespan in aging mice.

Effect of Selective Dorsal Neurectomy on Erectile Function in Rats

This study aimed to investigate the effect of penile selective dorsal neurectomy (SDN) on erectile function in rats. Twelve adult male Sprague-Dawley rats (15 weeks old) were divided into three groups (n=4 per group): in control group, rats received no treatment; in sham group, rats underwent a sham operation; in SDN group, rats underwent SDN with half of the dorsal penile nerve severed. The mating test was performed, and the intracavernous pressure (ICP) assessed six weeks after the surgical treatment. At postoperative six weeks, the mating test revealed no significant difference in mounting latency and mounting frequency among the three groups (P>0.05), while the ejaculation latency (EL) was significantly longer and ejaculation frequency (EF) lower in the SDN group than in the control and sham groups (P<0.05). There were no significant differences in preoperative and postoperative ICP and ICP/mean arterial blood pressure (MAP) among the three groups (P>0.05). SDN does not adversely affect the erectile function and sexual desire of rats, and at the same time it can reduce EL and EF, providing an application basis for SDN in the clinical treatment of premature ejaculation.

Ecological speciation of Japanese hedgehog mushroom: Hydnum subalpinum sp. nov. is distinguished from its sister species H. repando-orientale by means of integrative taxonomy

Hydnum repando-orientale is an East Asian species closely related to H. boreorepandum and H. repandum; all three species produce edible mushrooms. We identified two ecological groups of H. repando-orientale in Japan: a temperate group occurring in Fagaceae-dominated forest at < 1200 m a.s.l. (ROF) and a subalpine group occurring in coniferous forest in highland at > 1900 m a.s.l. (ROC). We re-examined the taxonomy of the two ecological groups of H. repando-orientale using integrative approaches. Phylogenies of the two ecological groups and other related species were inferred from the internal transcribed spacer (ITS) and gene portions encoding the large subunit of nc rRNA (LSU), translation elongation factor-1 alpha (TEF1), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second-largest subunit (RPB2). The concatenated phylogenetic tree separated the two ecological groups into well-supported sister clades. Also, species delimitations based on the topological congruence (GCPSR) and coalescent models (GMYC and BP&P) supported to separate the two ecological groups. Morphological analysis showed that ROC specimens had significantly larger basidiospores, compared with ROF specimens. Mon-mon mating tests using six ROF, three ROC, and three H. boreorepandum strains each showed independent incompatible groups, whereas one ROC strain showed compatibility with both ROC and ROF populations. Based on these results, we defined the ROC group as a new species, H. subalpinum. Because H. repando-orientale and H. subalpinum have smaller genetic divergence in nc rDNA and maintain slight sexual compatibility, they may have recently speciated in East Asia.

Paced Mating Behaviour Is Influenced by Duration of Female Post-Ejaculatory Interval

Laboratory paradigms that enable the female rat to control the frequency and temporal distribution of sexual stimulation are well-suited to gaining knowledge about female sexual function; however, the variety of procedures used influence the specific behaviors exhibited by female rats and bring uncertainty into the conclusions that can be drawn. In this study, we evaluated the effects of test parameters on the display of paced mating behavior in female rats to develop better preclinical models for exploring female sexual health. Sexually experienced, estradiol- and progesterone-primed female rats were tested under paced mating conditions to determine whether sexual behavior differed as a function of number of male partners (1 or 3; Experiment 1), the time span between receipt of an ejaculation and the next intromission (ie, the post-ejaculatory interval or PEI; Experiment 2), or the duration of ejaculations (Experiment 3). Contact-return latency, exit latency, and withdrawal duration after mounts, intromissions, and ejaculations. The shorter withdrawal latency after intromission and longer return latency after ejaculation observed in 30-minute paced mating tests is best attributed to the female's PEI. The duration of the PEI is a specific factor that affects the display of paced mating behavior. Understanding of neurobiological mechanisms and sensory factors influencing sexual behavior learned from these models can be applied to studies of human female sexual health. Sexual motivation is indicated by analogous behaviors and supported by evolutionarily conserved systems in women and rats, meaning that animal models can be used to identify neurobiological mechanisms subserving these behaviors. Nonetheless, data related specifically to physiological effects of different mating paradigms are needed in future research. Procedures that mimic naturalistic settings, and thus enable female sexual motivation to drive behavior, are apt for studies aimed at understanding mechanisms supporting female sexual function whereas artificially lengthening the interval between an ejaculation and subsequent intromission may provide a model to study female sexual dysfunction.

Taurine and its transporter TAUT positively affect male reproduction and early embryo development.

STUDY QUESTIONAre taurine and its transporter TAUT associated with spermiogenesis and early embryo development?SUMMARY ANSWERMorphologically abnormal spermatozoa increased after local functional interference by intratesticular injection, and taurine depletion significantly reduced the normal embryo numbers in vivo and blastocyst formation rate in vitro.WHAT IS KNOWN ALREADYTaurine is one of the most abundant amino acids in the male reproductive system and it has been demonstrated that taurine can efficiently improve spermatogenic function in rat models of testicular injury. However, limited information is known about the role of taurine and its transporter TAUT in spermatogenesis and early embryo development.STUDY DESIGN, SIZE, DURATIONClinical characteristics from 110 couples who have experienced recurrent pregnancy loss (RPL) were collected from December 2014 to March 2018. According to whether a fetal heartbeat was seen in the previous pregnancy under ultrasonic monitoring, patients with RPL were divided into two groups: an RPL without heartbeat (pregnancy with no fetal heartbeat, ROH) group, and an RPL with heartbeat (one or more pregnancies with fetal heartbeat, RWH) group. Semen samples (21 ROH and 20 RWH) were finally used for metabolomic analysis. Furthermore, semen samples were obtained from 30 patients with teratozoospermia (normal sperm morphology <4%) seeking evaluation for infertility and 25 age-matched control subjects with normal semen quality for western blotting. Animal experiments were performed in CD-1/ICR mice.PARTICIPANTS/MATERIALS, SETTING, METHODSMetabolomics was performed to determine the metabolic changes between the ROH and RWH groups. Sperm proteins from patients with teratozoospermia and healthy controls were extracted for detecting TAUT expression using western blot analysis. Immunofluorescence was used to characterize the localization of TAUT in the testis and ejaculated spermatozoa. Functional analysis in mice was performed by intratesticular injection of siRNAs or antagonist (β-alanine) and 5% β-alanine was provided in drinking water to 3-week-old male mice for 5 weeks with the aim of depleting taurine. Murine epididymal spermatozoa were stained with hematoxylin and eosin for morphological assessment. IVF and mating tests were performed in mice for assessing fertility.MAIN RESULTS AND THE ROLE OF CHANCEMetabolomic analysis demonstrated that the taurine content was lower in spermatozoa but higher in seminal plasma from the ROH than the RWH group. TAUT expression was lower in spermatozoa from patients with teratozoospermia than controls. Immunofluorescence showed that TAUT was localized to the manchette in mouse elongated spermatids functional analysis showed that morphologically abnormal spermatozoa increased after interference, and this defect increased after supplementation with 5% β-alanine but was improved by 5% taurine supplementation. Supplementation with 5% β-alanine significantly reduced the normal embryo number in the mouse uterus as well as blastocyst formation rate in vitro.LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONThe sample size was low and larger cohorts are needed to confirm the positive effect of taurine on human sperm quality. A comprehensive safety examination should be performed to evaluate whether taurine is a possible treatment for teratozoospermia. Furthermore, the specific molecular mechanism of TAUT involvement in spermiogenesis remains to be clarified.WIDER IMPLICATIONS OF THE FINDINGSThe study provides new insights into the role of taurine and its transporter TAUT in male reproduction and embryo development. The results also indicate that TAUT is a promising molecular candidate for the assessment of sperm quality, which may contribute to the diagnosis and treatment for teratozoospermia.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by grants from the National Natural Science Foundation of China (no. 81774075, 31900605, 81971451), Jiangsu Science and Technology Program Grant (BK20190654) and Maternal and child health scientific research of Jiangsu Province (F202121). The authors declare no competing financial interests.

Suppression of fertility by &lt;em&gt;Capparis spinosa&lt;/em&gt; root extract through antioxidant alteration in reproductive system of male rats

Aim: To evaluate the suppression of fertility by oral mode of Capparis spinosa root extract through alteration in antioxidant defence of the reproductive system of male albino rats. Methods: The methanol extract of Capparis spinosa root (100mg/kg/day) was fed for 60 days to male, Wistar strain, albino rats. The fertility and testicular functions were assessed by mating tests, antioxidant defence indices, and spermatogenesis, histometry, and biochemical estimations for fertility suppression along with serum biochemistry and hematology for assessment of toxicology. Results: Capparis spinosa root extract administration for a period of 60 days did not cause body weight loss significantly, while the weight of testes, Cauda epididymis, seminal vesicle, and ventral prostate were significantly reduced. The lipid peroxidation and antioxidants levels i.e. superoxide dismutase, catalase, glutathione reduced, glutathione peroxidase, vitamin C, and Vitamin E in testes, Cauda epididymis, seminal vesicle, and ventral prostate were altered significantly (P<0.001) after the 60 days treatment of Capparis spinosa extract when compared to the controls. The root extract feeding caused a marked reduction in the number of primary spermatocytes, secondary spermatocytes, and spermatids however spermatogonia population showed non-significant change. Sertoli cell population was also affected. The number of mature Leydig cells was decreased and degenerating cells increased proportionately. The seminiferous tubule and Leydig cell nuclear area were reduced significantly (P<0.001) when compared to the controls. The protein and sialic acid content of the testes, Cauda epididymis, seminal vesicle, and ventral prostate were significantly (P<0.001) decreased. The glycogen in testes and fructose in seminal vesicle was also significantly reduced, whereas the testicular cholesterol was elevated as compared with the controls. Reduced sperm count and motility resulted in a total suppression of fertility. The RBC and WBC count, hemoglobin, and hematocrit in whole blood whereas marker enzymes and glucose in serum were found to be within the normal range after the 60 days of Capparis spinosa extract treatment. Conclusion: Capparis spinosa root extract may selectively act on developing germ cells mediated via Sertoli cells through increased oxidative stress or impairment of antioxidant defence system in reproductive organs of male rats, leading to suppression of fertility and suggests the feasibility of developing non-toxic, herbal male contraceptive drug.

Systematic revision of Hydnum species in Japan

ABSTRACT Hydnum (Hydnaceae, Basidiomycota) exhibits endemic species diversity in East Asia; however, few comprehensive systematic studies have been conducted to date. Here, we performed morphological, ecological, phylogenetic, and biological evaluations of the taxonomy of Hydnum species in Japan. In total, 186 Japanese Hydnum specimens were used for morphological observations. Phylogenetic trees were constructed using sequence data of nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) region and a portion of translation elongation factor 1-α (tef1). Intra- and interspecific mating tests using 78 monokaryotic strains of 13 species did not conflict with species delimitation inferred from their ITS and tef1 phylogenetic relationships. This study provides detailed morphological descriptions of 15 rigorously identified species from Japan, nine of which are described as new: H. alboluteum, H. albopallidum, H. pinicola, H. itachiharitake, H. minospororufescens, H. orientalbidum, H. subberkeleyanum, H. tomaense, and H. tottoriense. Three species documented in this work are new to Japan: H. boreorepandum, H. mulsicolor, and H. umbilicatum. The remaining three species (H. cremeoalbum, H. minus, and H. repando-orientale), previously reported from Japan, are redescribed using data from newly collected materials. We also transferred two old species (Hericium fimbrillatum and Sarcodon nauseofoetidus) from East Asian Hydnum into other genera.

Isolating Caenorhabditis elegans from the Natural Habitat.

Wild populations of the model organism C. elegans represent a valuable resource, allowing for genetic characterization underlying natural phenotypic variation. Here we provide a simple protocol on how to sample and rapidly identify C. elegans wild isolates. We outline how to find suitable habitats and organic substrates, followed by describing isolation and identification of C. elegans live cultures based on easily recognizable morphological characteristics, molecular barcodes, and mating tests. This protocol uses standard laboratory equipment and requires little prior knowledge of C. elegans biology.

Effect of Combined Oyster Peptide and Ginseng Extracts on Improvement of Sexual Function and Anti-fatigue

This study aimed to explore the effects of the combination of oyster peptide and ginseng extracts (OPGE) on exercise fatigue and sexual function in normal male mice. In this experiment, ICR male mice were randomly divided into 6 groups, including control group, oyster peptide (0.2 mg/gbw, OP), ginseng extract (0.2 mg/gbw, GE), low dosage of OPGE (0.1 mg/gbw, OPGEL), medium dosage of OPGE (0.2 mg/gbw, OPGEM) and high dosage of OPGE (0.6 mg/gbw, OPGEH). All animals were treated continuously by oral gavage for 42 days, and then the swimming experiment and mating test were respectively performed on the last day of administration. Anti-fatigue and sexual function-related biochemical parameters were measured. The mating ability of mice was significantly enhanced in each treatment group compared with control group. The mating ability of OP and GE treated alone showed inferior to OPGEM administration, but without statistical difference. Compared with the control group, the serum levels of testosterone and follicle-stimulating hormone, the nitric oxide level in penis was increased by 20.02%, 19.92% and 17.19% respectively, while the content of phosphodiesterases-5 in penis was decreased in OPGEM group. The result indicated that OPGEM administration presented the best effect on the promotion of sexual function among all groups (P 0.05). Furthermore, the levels of lactic acid(P<0.01), urea nitrogen (P<0.05) and malondialdehyde(P<0.01) were decreased by the administration of OPGEM, while the content of glycogen was increased (P<0.01). In conclusion, OPGE presented a synergistic effect on anti-fatigue and improvement of sexual ability in normal male mice.The results are beneficial to promote the development of the functional food industry in the related field.

Epigallocatechin gallate and theaflavins independently alleviate cyclophosphamide-induced ovarian damage by inhibiting the overactivation of primordial follicles and follicular atresia

BackgroundCyclophosphamide (CTX), which has been used to treat common female cancers for several years, often causes ovarian damage, early menopause and infertility. However, strategies for the effective prevention and treatment of CTX-induced ovarian damage are still lacking. Epigallocatechin gallate (EGCG) and theaflavins (TFs), key molecules derived from green tea or black tea, have been shown to exert preventive effects on many ageing-related diseases. PurposeWe aimed to explore the potential preventive and protective effects of EGCG and TFs on CTX-induced ovarian damage and compare the two compounds. Study DesignSix-week-old female mice were administered a low or high dose of EGCG or TFs. The low dose was equivalent to the average daily amount of tea consumed by a drinker. MethodsWe determined the oestrous cycle and serum hormone levels to evaluate ovarian endocrine function, and we performed mating tests for reproductivity. We also assessed the follicle count and AMH level to evaluate ovarian reserve, and we performed Masson's trichrome and Sirius red staining to evaluate ovarian fibrosis. We conducted γ-H2AX and TUNEL analyses to evaluate DNA damage, and we also measured the relevant indicators of oxidative stress and follicular activation, including NRF2, HO-1, SOD2, AKT, mTOR and RPS6. ResultsEGCG and TFs treatment independently improved the ovarian endocrine function and reproductivity of mice that were administered CTX. EGCG and TFs also increased the ovarian reserve of these animals. Furthermore, EGCG and TFs alleviated oxidation-induced damage to ovarian DNA in mice by activating the NRF2/HO-1 and SOD2 pathways and reducing the apoptosis of growing follicles. At the same time, EGCG and TFs reduced the overactivation of primordial follicles by inhibiting the AKT/mTOR/RPS6 pathway. ConclusionThe present study showed that EGCG and TFs independently improved ovarian function in mice with CTX-induced ovarian damage, thereby providing useful information for designing a potential clinical strategy that will protect against chemotherapy-induced ovarian damage.

P–036 Investigation of the 2100 MHz electromagnetic field effects on sperm CatSper calcium channel

Abstract Study question Does electromagnetic field (EMF) effect sperm motility through CatSper calcium channels in rat? Summary answer 2100 MHz EMF may reduce sperm motility by acting on CatSper calcium channels. What is known already EMF exposure has become a serious concern in infertility patients. The effects of EMF through by using mobile phone and laptop have been explored previously, mostly focusing on sperm motility and DNA fragmentation. EMF activates the voltage gated calcium channels and increases calcium concentration. As a result of the EMA exposure, the sperm motility may increase. However, if this happens while sperms are in non-progressive motile phase in the epididymis, it may result with the depletion of limited energy stores. Sperms may become immotile and they can’t move forward in the progressive motile phase in the female reproductive system. Study design, size, duration This basic research study was an in vivo experimental approach involving the use of 50 male rats. Wistar-Albino rats (n = 10) weighing ∼320 –350g were included in each groups. The duration of EMF exposure was 1 hour per day for 28 days. Amlodipine (1 mg/kg, 28 days) was used as a calcium channel blocker. The experiment was held between July to December in 2020. 20 female rats were recruited for mating test. Participants/materials, setting, methods 50 rats were divided into five groups. Group 1; Pure control. Group 2; Sham. Group 3; EMF exposure, Group 4: EMF+Amlodipine, Group 5: Amlodipine positive control. After four weeks of exposure, rats were sacrificed and sperm were collected from cauda epididymis. Sperm motility parameters were analyzed. Intracellular calcium levels were determined with two different method, fluorescence spectrophotometer and laser scanning confocal microscope. Before sacrifice, rats were mated with female rats to evaluate mating ratios. Main results and the role of chance The mating score and live birth rates did not vary significantly among the groups (p &amp;gt; 0.05). The sperm motility (A+B, 47.62±16.92 versus 34.19±14.62) and intracellular calcium levels (2.46±0.20 versus 1.85±0.18) were significantly decreased in the EMF group (p &amp;lt; 0.05). The results of fluorescence spectrophotometer and laser scanning confocal microscopy with fluorescent attachment were consistent with each other. There were no significant differences found among the other groups in terms of investigated parameters. Statistical analysis was performed with Kruskal-Wallis test followed by the Dunn-Bonferroni’s test. Limitations, reasons for caution The Catsper 1, 2, 3, 4 gene expression levels are still under analyses. These gene expression levels will be helpful to understand possible changes of the sperm motility. The determination of other motility related gene expressions may strength the results. Wider implications of the findings: EMF exposure may have a significant effect on sperm motility parameters. Mobile phones carried very close to the reproductive organs may adversely affect the motility of sperm cells due to its emitted radiation levels Trial registration number Not applicable