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Problem The aim of this study was to find immune-related genes expressed in cumulus cells of ovulated cumulus oocyte complexes ( COCs) and to clear the functional roles during fertilization process. Method of study Ovulated COCs were collected from oviduct 16 hr after the hCG injections followed by eCG priming. The cumulus cells were used for RT-PCR or western blotting study. COCs were also used for in vitro fertilization study. Results Cramp, Trf, Lyz2, S100a8, and S100a9 were expressed in cumulus cells during ovulation process. The protein levels of CRAMP or transferrin were detected in ovulated COCs and then secreted into hyaluronan-rich matrix. The high dose of these factors reduced the proliferative activity of E. coli; however, the lower levels of them significantly increased the rate of fertilization in in vitro via the induction of sperm capacitation. Conclusion Cumulus-secreted anti-bacterial factors act on sperm to induce sperm capacitation. [ABSTRACT FROM AUTHOR]

How does a regime change influence elite mobility? By collecting data on elites after the Meiji Restoration in Japan in 1868, through which Japan transitioned from a feudal regime to a modern regime, we provide new evidence that the impact of the regime change on elite mobility varies across the stages of the regime change. We analyze the impact of the regime change from two aspects: (1) the composition of elites or elite membership and (2) the internal hierarchy within them. The regime change opened an opportunity for commoners to join the elite group. After the Meiji Restoration, the share of elites whose fathers were commoners in the former regime increased, as did the influence of meritocracy on elite ranks. However, once the new regime was established, the elite hierarchy started to reflect the social stratum of the former regime and the influence of meritocracy declined., (© 2023 The Authors. The British Journal of Sociology published by John Wiley & Sons Ltd on behalf of London School of Economics and Political Science.)

Sperm motility patterns are continuously changed after ejaculation to fertilization in the female tract. Hyperactivated motility is induced with high glucose medium in vitro or the oviduct fluids in vivo , whereas sperm maintain linear motility in the seminal plasma or the uterine fluids containing low glucose. Therefore, it is estimated that sperm motility patterns are dependent on the energy sources, and the mitochondrial oxidative phosphorylation is activated to produce ATP in low glucose condition. To elucidate these hypotheses, boar sperm was incubated in different energy conditions with the transcription and translation inhibitors in vitro . Sperm motility parameters, mitochondrial activity, ATP level, gene expression and protein synthesis were analyzed. Sperm progressive motility and straight-line velocity were significantly increased with decreasing glucose level in the incubation medium. Moreover, the mitochondrial protein turnover meaning transcription and translation from mitochondrial genome in sperm is activated during incubation. Incubation of sperm with mitochondrial translation inhibitor (D-chloramphenicol) suppressed mitochondrial protein synthesis, mitochondrial activity and ATP level in sperm and consequently reduced the linear motility speed, but not the motility. Thus, it is revealed that the mitochondrial central dogma is active in sperm, and the high-speed linear motility is induced in low glucose condition via activating the mitochondrial activity for ATP generation.

Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co-injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen-thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen-thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen-thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen-thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance., (© 2013 Japanese Society of Animal Science.)

Frozen-thawed epididymal spermatozoa have good fertilization capability in vitro; however, their artificial insemination conception rate is less than half of that of frozen-thawed ejaculated spermatozoa. Because the addition of seminal plasma to the thawing solution enhances the in vivo fertilizing ability of frozen-thawed ejaculated spermatozoa, we hypothesized that the reproductive performance of frozen-thawed epididymal spermatozoa could also be improved by the inclusion of seminal plasma. When frozenthawed epididymal spermatozoa were incubated for up to 6 hours, the motility of the sperm significantly decreased in a time-dependent manner. The acrosomal membrane was damaged in the majority of frozen-thawed epididymal spermatozoa. The addition of seminal plasma to the thawing solution significantly decreased the percentage of sperm with abnormal acrosomes and increased their total motility in a dose-dependent manner. Furthermore, the addition of seminal plasma reduced the abundance of a 15-kDa tyrosinephosphorylated protein in frozen-thawed sperm, and the maximum effect was observed at 15% (vol/vol) seminal plasma. When cryopreserved epididymal spermatozoa from 3 different boars were thawed with a 15% (vol/vol) seminal plasma-containing solution, the conception rate and mean litter size obtained by artificial insemination were significantly increased as compared with those in the control without seminal plasma. From these results, we concluded that the addition of seminal plasma to the thawing solution is a key step in obtaining an optimal number of piglets by artificial insemination using frozen-thawed boar epididymal spermatozoa.

Cryopreservation of boar spermatozoa offers an effective means of long-term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70-80%) can be achieved by AI with frozen-thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria-released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll-like receptor-4 (TLR-4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo-capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen-thawed boar sperm., (© 2012 Japanese Society of Animal Science.)

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation-like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal-derived materials. In this study, we focused on the increase of intracellular Ca(2+) ([Ca(2+)](i)) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca(2+)](i) indicator, Fluo-3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca(2+) chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post-thawed sperm motility. When the frozen-thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca(2+)](i) and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen-thawed boar sperm., (© 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.)

Gram negative bacteria are the predominant type detected in boar semen. Since these bacteria release lipopolysaccharide (LPS), and because polymyxin B (PMB) neutralizes LPS activity, the objective was to improve techniques for long-term storage of boar sperm by testing the beneficial effects of PMB. In the present study, LPS bound directly to the head region of sperm, decreased sperm motility, and induced sperm apoptosis. The addition of 100 μg/mL PMB suppressed LPS binding activity and blocked the negative effects of LPS on sperm quality. Additionally, when PMB treatment was combined with penicillin G (PenG), sperm motility was increased after 10 d of liquid storage or in frozen-thawed sperm (P<0.05). When the PMB- and PenG-treated sperm was used for artificial insemination, the conception rate was increased relative to that of artificial insemination with sperm treated by PenG alone in both liquid (62 vs. 81%) and cryopreserved forms (50 vs. 80%, P < 0.05). We concluded that PMB suppressed LPS-induced low sperm motility and apoptosis via the reduction of LPS binding to Toll-like receptors (TLRs). Thus, in order to enhance sperm quality for artificial insemination, a combined treatment with PMB and PenG immediately after ejaculation seemed appropriate to maintain sperm motility and function during both liquid storage and cryopreservation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.

The present study aimed to investigate progesterone receptor (PR) gene expression in cumulus cells and their roles during meiotic resumption of porcine oocytes. The amount of PR-A or PR-B mRNA was analyzed by RT-PCR using primer sets for the PR-B region or the PR-A/B common region. The level of PR-B mRNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation but the level significantly decreased at 12 h. However, a high level of total PR mRNA was maintained up to a cultivation period of 20 h. The level of PR-B protein in cumulus cells reached its maximum at 4 to 12 h, whereas PR-A predominated in cumulus cells of cumulus-oocyte complexes (COCs) at 20 h. Accompanying the shift in expression of PR isoforms, progesterone production in cumulus cells was significantly increased, and both the proliferative activity of cumulus cells during a 10- to 20-h cultivation period and the level of connexin-43, a major component of the gap junction, in cumulus cells significantly decreased. When COCs were cultured with FSH and LH for 10 h and then further cultured with additional RU486, there was a significant suppression in the shift in PR isoforms and in progesterone production, a loss of proliferative activity, and a decrease in connexin-43 mRNA in cumulus cells. Moreover, treatment with RU486 after 10-h cultivation of COCs inhibited the meiotic resumption of oocytes and cumulus cell expansion. These results suggest that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to impact on proliferation and differentiation in a time-dependent manner, and the shift from PR-B to PR-A may help mediate certain events.

It has been reported that LH receptor (LHR) mRNA is not detected in cumulus cells of porcine cumulus-oocyte complexes (COCs) just after collection from small antral follicles. The present study showed that the formation of LHR in cumulus cells was up-regulated by the cultivation with 20 ng/ml FSH. When the newly synthesized receptors were stimulated by 1.0 microg/ml LH, significantly higher levels of cAMP and progesterone production in cumulus cells were observed as compared with those of COCs cultured with FSH. A loss of proliferative activity of cumulus cells was induced by the additional LH to FSH-containing medium; however, the inhibitory effect was overcome by progesterone receptor antagonist RU486. Furthermore, the addition of LH also accelerated ongoing GVBD in cumulus cells-enclosed oocytes. These results revealed that during in vitro meiotic maturation of porcine COCs, progesterone secreted by FSH- and LH-stimulated cumulus cells reduced proliferative activity of cumulus cells; the changes of cumulus cells might be involved in inducing meiotic resumption of porcine oocytes.