Expression of Relaxin Family Peptide Receptors 1 and 3 in the Ovarian Follicle of Japanese Quail.

In our previous studies, we demonstrated that the primary source of relaxin 3 (RLN3) in Japanese quail is ovarian granulosa cells. Although several relaxin family peptide (RXFP) receptors have been sequenced, the intricacies of these receptors in avian species remain insufficiently clarified. Therefore, we assessed the expression of RXFP receptors, RXFP1 and 3, in Japanese quail. Using RT-PCR, we found that both RXFP1 and 3 were ubiquitously expressed. The expression level of RXFP1 is significantly higher in the ovarian theca layer, indicating that it is the primary receptor for RLN3 in the ovary. During follicular development, there was an elevation in thecal RXFP1 expression, but it declined after the luteinizing hormone (LH) surge. We found that the protease activity of the 60 kDa band increased after the LH surge, suggesting the involvement of RLN3 signaling in ovulation. These results suggest a paracrine role of RLN3, involving its binding with RXFP1 in ovarian theca cells. This interaction may elicit biological actions, potentially initiating ovulation after the LH surge.

Expression of Relaxin 3 in the Ovarian Follicle of Japanese Quail.

The relaxin (RLN) gene is expressed in the reproductive tracts, such as the ovary and uterus, of mammalian species. Although RLN expression is detected in the chicken ovary, detailed clarification of the physiological role of RLN has not yet been reported. To address this issue, in the present study we aimed to examine the spatiotemporal expression and hormonal control of RLN in Japanese quail. By performing semi-quantitative and quantitative reverse transcription-polymerase chain reaction analysis, we found that RLN mRNA was mainly expressed in the granulosa and theca layers of the ovary. The expression level in the granulosa layer increased with the stage of follicular development. Results from granulosa layer culture experiments revealed that RLN mRNA expression increased with the addition of estradiol-17ß, whereas the addition of progesterone suppressed RLN transcription. More detailed analysis indicated that RLN expression was highest in the stigma region of the follicle but significantly decreased as the time of the expected luteinizing hormone (LH) surge approached. Together, our findings demonstrated that the granulosa cells in the mature preovulatory follicles constitute the main source of RLN in the Japanese quail. Because RLN expression was highest in the stigma region and the expression dramatically decreased following the LH surge, the results further suggest that RLN may be related to tissue remodeling for the ovulation process in birds.

Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing.

Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.

Egg Development After In Vitro Insemination in Japanese Quail (Coturnix japonica).

In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca2+ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 104). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 104 is required for the progression of the Ca2+ signaling pathway, which initiates subsequent egg development in Japanese quail.

Regulation of Prolactin Release at the End Stage of Chicken Embryogenesis.

Difference of onset of increase of PRL content in the anterior pituitary gland and plasma PRL concentration during the late stage of chicken embryogenesis is well known. To investigate the disagreement, changes in PRL content and PRL mRNA levels, and the effects of vasoactive intestinal polypeptides (VIP) on PRL release and PRL mRNA expression were examined using western blot analysis and real-time PCR quantification. Changes in SPRL content were strongly correlated with PRL mRNA levels. The increase in PRL content on day 17 of incubation may be caused by the increase in PRL mRNA levels on day 16 of incubation. Additionally, the effects of VIP on PRL release from the embryonic anterior pituitary gland were not observed until day 18 of embryogenesis. These results suggest that increased levels of PRL mRNA and PRL content in the anterior pituitary gland are closely correlated. However, the increased expression of PRL mRNA observed on day 17 and the initiation of PRL release from the anterior pituitary gland on day 19 were differentially regulated. According to the results of western blot analysis, the proportion of glycosylated PRL (G-PRL) and non-glycosylated PRL (NG-PRL) in the anterior pituitary gland at the end stage of development differed from the proportion of PRL released from the anterior pituitary gland. According to the results of two-dimensional western blot analysis, no isoforms with different isoelectric points were detected in the culture medium on days 19 and 20. These data suggest that the peptide chains of G-PRL and NG-PRL were not modified. In conclusion, the differentiation of PRL-producing cells and the maturation of the hypothalamus and anterior pituitary gland were completed at the end stage of incubation, and that different factors regulated the initiation of PRL mRNA expression before day 18 of incubation.

Possible involvement of annexin A6 in preferential sperm penetration in the germinal disk region.

Abstract: During fertilization, avian sperm preferentially penetrate into the perivitelline membrane that covers the germinal disk region where the female nucleus is present. This phenomenon has been observed not only in domestic birds but also in wild birds; however, the mechanisms controlling sperm preference are still unclear. In this study, we investigated the possible involvement of annexin family protein in sperm-egg interaction in Japanese quail. Microscopic examination of fertilized eggs indicated that quail sperm penetration only occurred in the germinal disk region, and sperm localized outside the germinal disk were trapped in the perivitelline membrane. Western blot analysis and immunofluorescence microscopy revealed the presence of annexin A1 and A6 in the oocyte membrane, while annexin A6 localized in the perivitelline space of the germinal disk region. Further, our sperm binding assay using recombinant annexin A6 demonstrated that ejaculated sperm specifically bound to annexin A6 expressed in mammalian cell lines. These results suggest that annexin A6, which is expressed on the surface of oocytes, may function in sperm-egg interaction in the germinal disk region and that this binding may ensure sperm retention on the surface of the egg plasma membrane until fertilization takes place in Japanese quail. Lay summary: In bird species, fertilization takes place immediately after ovulation of the egg. Sperm preferentially penetrate a specific area of the egg coating that covers the 'germinal disk region' - this area contains the cell that needs to be fertilized by a sperm. However, since the bird egg is extremely large in size and sperm must reach the 'germinal disk region' to achieve fertilization, it is unclear how this happens. Annexin proteins support fertilization in mammals, and we found that annexin A6 protein exhibits a unique localization in the germinal disk region in the eggs of Japanese quail. To test this interaction, we incubated quail sperm with cells that produced annexin A6 and found that ejaculated sperm bound to the cells. These results suggest that annexin A6 may have a role in the sperm-egg interaction in the germinal disk region in Japanese quail.

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca2+ ([Ca2+]i): transient elevations in [Ca2+]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca2+ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca2+]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca2+ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca2+]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca2+ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca2+]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca2+ wave and spiral-like Ca2+ oscillations, respectively.

Transgenerational effects of bisphenol A on zebrafish reproductive tissues and sperm motility.

In a previous study, we demonstrated the next-generation effects and further transgenerational adverse effects of bisphenol A (BPA) in zebrafish. The adverse effects on reproductive factors, such as gonadal activity, fertility, hatching rate and malformation of embryo caused by the dietary administration on initial generation (F0) male and female zebrafish were continued until third filial (F3) generation. In this study, we examined how much amount of BPA contained in the diet was taken up by the zebrafish. We showed that only about 3.5-6.8% of BPA in the diet was taken into fish body. Also, we confirmed the transgenerational effects caused by 100 times lower amount of BPA than previous study. Even a low amount of BPA (1 µg/g diet) administered to F0 not only caused retraction of the ovaries and testes but also lowered the survival rate and increased the rate of malformation in the offspring. The effects were continued to F3 generation as previously described. Moreover, the sperm motility of the offspring of the BPA-treated ancestral animals was significantly lower, and this adverse effect was continued to F2 generations. These findings demonstrated that BPA at levels comparable to those ingested by humans can cause transgenerational adverse effects on fish reproduction.

Sperm Motility Regulation in Male and Female Bird Genital Tracts.

Sperm drastically change their flagellar movement in response to the surrounding physical and chemical environment. Testicular sperm are immotile; however, they gain the competence to initiate motility during passage through the male reproductive tract. Once ejaculated, the sperm are activated and promptly initiate motility. Unlike mammals, ejaculated sperm in birds are stored in specialized tubular invaginations referred to as sperm storage tubules (SSTs), located between the vagina and uterus, before fertilization. The resident sperm in the SSTs are in a quiescent state and then re-activated after release from the SSTs. It is thought that avian sperm can undergo motility change from quiescent to active state twice; however, the molecular mechanism underlying sperm motility regulation is poorly understood. In this short review, we summarize the current understanding of sperm motility regulation in male and female bird reproductive tracts. We also describe signal transduction, which regulates sperm motility, mainly derived from in vitro studies.

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility, free thiol content and seasonality.

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.

Effect of Anserine and Carnosine on Sperm Motility in the Japanese Quail.

Sperm motility is considered as one of the most important traits for successful fertilization, but the motility of an ejaculated sperm decreases with time when stored as liquid. It is reported that seminal plasma serves as a nutrient rich medium for sperm and plays an important role in sperm motility and its fertilization ability. Several studies have reported that imidazole dipeptides such as anserine and carnosine affect sperm motility and its fertilization ability in mammals. In this study, we report the presence of anserine and carnosine in the male reproductive tract of the Japanese quail. Abundant levels of anserine (44.46 µM) and carnosine (41.75 µM) were detected in the testicular fluid and seminal plasma respectively using the amino acid analyzer; however, seminal plasma solely contained carnosine. When the ejaculates were incubated with anserine or carnosine, we found that both the dipeptides improve sperm motility parameters such as straight line velocity, curvilinear velocity, average path velocity and amplitude of lateral head displacement after in vitro sperm storage at 15°C. These results indicate that imidazole dipeptides are present in the male reproductive tract and may improve sperm quality during in vitro sperm storage in the liquid states.

Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail.

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

Effect of sperm surface oligosaccharides in sperm passage into sperm storage tubules in Japanese quail (Coturnix japonica).

In birds, the ejaculated spermatozoa do not directly pass to the site of fertilization but rather are stored initially in specialized structures, referred to as sperm storage tubules (SSTs), located in the utero-vaginal junction (UVJ) of the oviduct. The fertilizing capacity of spermatozoa in the SSTs is maintained for an extended period (i.e., several days to months). Although many studies have been conducted to ascertain the mechanisms involved in sperm storage, the understanding of the phenomenon is limited. In this study, there was investigation of the effects of sperm surface oligosaccharides in sperm passage into SSTs in Japanese quail. Results from lectin staining of ejaculated spermatozoa indicated galactose/N-Acetylgalactosamine (Gal/GalNAc), N-Acetylglucosamine (GlcNAc) or mannose/glucose (Man/Glc) moieties were present on the sperm surface, indicating the presence of glycoproteins/glycolipids containing these oligosaccharides. When ejaculated spermatozoa were co-incubated with UVJ explants, the lectins derived from Agaricus bisporus and Canavalia ensiformis had marked inhibitory effects on sperm passage into SSTs. Preincubation of UVJ explants with these lectins, however, had no effect indicating there were no effects of UVJ oligosaccharides in this process. Furthermore, none of these lectin had effects on values of sperm motility variables. These results indicate that O-glycans with terminal ß-Gal or GalNAc and N-glycans with terminal α-D-Man or α-D-Glc may have functions in the process of sperm passage into SSTs.

Longer and faster sperm exhibit better fertilization success in Japanese quail.

In birds, sperm storage tubules (SST) located in the utero-vaginal junction are thought to be a site of sperm selection; however, the exact mechanism of sperm selection is poorly understood. Here, we investigated sperm entry into the SST and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from 2 males. We employed 2 quail strains, a wild-type and a dominant black (DB) type, as this allows easy assessment of paternity by feather coloration. We found paternity of embryos was biased toward DB males when a sperm mix with similar sperm numbers from the 2 males strains was artificially inseminated into females. Our novel sperm staining method with 2 different fluorescent dyes showed that the DB-biased fertilization was because of the better ability of DB sperm to enter the SST. Moreover, we found that DB sperm had a longer flagellum and midpiece. These characteristics probably allow sperm to swim faster in a high viscosity medium, which may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SST and that filling the SST with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail.

An efficient heterologous Escherichia coli-based expression system for lectin production from Pleurocybella porrigens.

In this study, we report a more efficient heterologous expression of lectin from Pleurocybella porrigens (PPL) using an Escherichia coli-based expression system. The yield (9.3 mg/L culture broth) of recombinant PPL (rPPL) using this expression system was increased approximately 9-fold compared to our previous study. The rPPL obtained in this study exhibited the same biochemical properties as the native PPL.

Rare polyandry and common monogamy in the firefly squid, Watasenia scintillans.

In cephalopods, all species are considered to be polyandrous because of their common life history and reproductive traits reflecting a polyandrous mating system. Contrary to this belief, here we show several lines of evidence for monogamy in the firefly squid, Watasenia scintillans. In this species, females are capable of long-term storage of spermatangia, and of egg spawning even after the complete disappearance of males following the breeding season. The stored spermatangia are distributed equally between bilateral pouches under the female's neck collar. Such a nonrandom pattern of sperm storage prompted us to hypothesize that females might engage in lifetime monandry. Hence, we genotyped female-stored spermatangia and offspring, and found that in 95% of females (18/19), all the spermatangia had been delivered from a single male and all the embryos in a clutch had been sired by spermatozoa from stored spermatangia. In males, throughout the reproductive season, relative testis mass was much smaller in W. scintillans than in all other cephalopods examined previously. The mean number of male-stored spermatophores was ~ 30, equivalent to only 2.5 matings. Our genetic, demographic and morphometrical data agree with a mathematical model predicting that monogyny is favored when potential mates are scarce. Together, these results suggest mutual monogamy in W. scintillans.

Expression of Transferrin and Albumin in the Sperm-Storage Tubules of Japanese Quail and their Possible Involvement in Long-Term Sperm Storage.

Because of the presence of sperm storage tubules (SSTs) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm enter the female reproductive tract, they can survive for a prolonged period in domestic birds; however, the specific mechanisms involved in sperm maintenance within the SST remain to be elucidated. In this study, we showed that transferrin (TF) and albumin (ALB) are expressed in SSTs. When UVJ extracts were subjected to size-exclusion column chromatography, we obtained fractions that extend sperm longevity in vitro. LC-MS/MS analysis of the two major proteins in the fractions identified these proteins as TF and ALB. Immunohistochemical analysis using specific antisera against TF and ALB indicated that both proteins were localized not only in the SSTs, but also in the surface epithelium of the UVJ. When the ejaculated sperm were incubated with either purified TF or ALB, sperm viability increased after 24 h. These results indicated that oviductal TF and ALB are involved in the process of sperm storage in SSTs and may open a new approach for technological improvement to prolong sperm longevity in vitro.

Efficacy of relaxin for cisplatin-induced testicular dysfunction and epididymal spermatotoxicity.

BACKGROUND: Cisplatin (CP) is an extremely effective anticancer agent widely used to treat various cancer types, however, the potential side effects include testicular dysfunction. This study was to investigate, using a rat model of CP-induced testicular dysfunction, the protective effects of relaxin (RLN) against oxidative stress, testicular function, histological damage, spermatogenesis, germ-cell apoptosis, and sperm output, and to explore the usefulness of RLN as a potential protective drug for use with CP in chemotherapeutic treatments. METHODS: Sprague-Dawley male rats were used, which were divided into three groups: sham control, CP, and CP + RLN. Porcine RLN (500 ng/h) or saline was infused for 5 days using an implanted osmotic mini-pump following intraperitoneal injection of CP (6 mg/kg). RLN dose was chosen based on previous studies showing that it resulted in serum relaxin levels comparable to those in rats at the middle of pregnancy. At 5 days after CP administration, samples were collected and assessment of testicular histopathology, germ-cell apoptosis, oxidative stress, lipid peroxidation, and sperm quality was performed as main measures. RESULTS: The testicular CP model showed reduced testis weight and significantly decreased spermatogenesis scores. Additionally, CP administration induced a 4.6-fold increase in the apoptotic index associated with a significant increase in oxidative stress and upregulation of pro-apoptotic Casp3 and downregulation of anti-apoptotic Bcl2 levels, resulting in a marked reduction in sperm concentration. However, RLN administration caused a significant reduction in CP-mediated damage by attenuating oxidative stress and cell apoptosis. RLN administration efficiently scavenged ROS via the activation of SOD, CAT, and GPx and upregulation of GSH to prevent lipid peroxidation and decreased apoptosis by altering Bcl2 and Casp3 expression, thereby reducing histopathological damage and restoring spermatogenesis. Furthermore, RLN ameliorated attenuated sperm motility in the cauda epididymis resulting from CP treatment. CONCLUSIONS: This study clearly indicates that RLN exerts a protective effect against CP-induced testicular damage through attenuation of oxidative stress and suppression of apoptosis. Our findings suggest RLN as a potentially efficacious drug for use with cisplatin chemotherapy in order to ameliorate CP-induced side effects and testicular injury adversely affecting spermatogenesis, sperm quality, and oxidative-stress parameters.

CONTEXTE: Le cis platine (CP) est un agent anticancéreux extrêmement efficace largement utilisé pour traiter divers types de cancer. Parmi les effets secondaires potentiels associés aux traitements par CP on compte le dysfonctionnement des testicules. Le propos de ce manuscrit est d'étudier, à l'aide d'un modèle rat de dysfonctionnement testiculaire induit par la prise de CP, l'action protectrice de la relaxine (RLN) contre les effets délétères dus au CP lesquels incluent le stress oxydant, la perte de fonction testiculaire, les dommages histologiques au testicule, l'apoptose des cellules germinales et la baisse de la qualité des spermatozoïdes. L'objectif est d'explorer l'utilité de la RLN comme médicament protecteur potentiel à utiliser avec le CP dans les traitements chimiothérapeutiques. MÉTHODES: Des rats mâles Sprague-Dawley ont été utilisés. Trois groupes : contrôle, CP, et CP + RLN ont été comparés. Après une injection intrapéritonéale de CP (6mg/kg), de la RLN porcine (500 ng/h) ou du sérum physiologique a été perfusé pendant 5 jours en utilisant une mini-pompe osmotique implantée. La dose de RLN a été choisie en fonction d'études antérieures qui avaient montré qu'elle entraînait des taux sériques de RLN comparables à ceux de rats en milieu de la gestation. Cinq jours après l'administration de la CP, des échantillons ont été prélevés afin d'évaluer l'histopathologie, l'apoptose des cellules germinales, le stress oxydant, la peroxydation des lipides et les paramètres spermatiques. RÉSULTATS: Le groupe CP a montré une réduction du poids des testicules et une diminution significative des scores de spermatogenèse. De plus, l'administration de CP a entraîné une augmentation de l'apoptose de 4,6 fois associée à une augmentation significative du stress oxydant, de la régulation à la hausse de la Caspase 3 pro-apoptotique et à la baisse de Bcl2 anti-apoptotique conduisant in fine à une réduction marquée de la concentration en spermatozoïdes. La RLN a ainsi significativement corrigée les effets négatifs du CP en atténuant le stress oxydant et l'apoptose. La RLN a permis d'éliminer efficacement les ROS via l'activation de la triade enzymatique anti-oxydante superoxyde dismutase (SOD)/catalase (CAT)/glutathion peroxydase (GPx) et via la régulation à la hausse du GSH prévenant ainsi la lipopéroxydation. La RLN a par ailleurs diminué les atteintes histopathologiques testiculaires préservant la spermatogenèse. En parallèle, la RLN a amélioré la mobilité spermatique des spermatozoïdes prélevés dans la queue de l'épididyme. CONCLUSIONS: Cette étude montre clairement que la RLN exerce un effet protecteur contre les lésions testiculaires par l'atténuation du stress oxydant et la suppression de l'apoptose induite par le CP. Nos résultats suggèrent que la RLN est un médicament potentiellement pertinent à utiliser afin de diminuer les effets secondaires induits par le CP sur la fonction testiculaire et sur les spermatozoïdes lors de la chimiothérapie cancéreuse.

Female Japanese quail visually differentiate testosterone-dependent male attractiveness for mating preferences.

Biased mating due to female preferences towards certain traits in males is a major mechanism driving sexual selection, and may constitute an important evolutionary force in organisms with sexual reproduction. In birds, although the role of male ornamentation, plumage coloration, genetic dissimilarity, and body size have on mate selection by females have been examined extensively, few studies have clarified exactly how these characteristics affect female mate preferences. Here, we show that testosterone (T)-dependent male attractiveness enhances female preference for males of a polygamous species, the Japanese quail. A significant positive correlation between female mating preference and circulating T in the male was observed. The cheek feathers of attractive males contained higher levels of melanin and were more brightly colored. The ability of females to distinguish attractive males from other males was negated when the light source was covered with a sharp cut filter (cutoff; < 640 nm). When females were maintained under short-day conditions, the expression of retinal red-sensitive opsin decreased dramatically and they became insensitive to male attractiveness. Our results showed that female preference in quail is strongly stimulated by male feather coloration in a T-dependent manner and that female birds develop a keen sense for this coloration due to upregulation of retinal red-sensitive opsin under breeding conditions.

Accelerated invagination of vacuoles as a stress response in chronically heat-stressed yeasts.

When exposed to sublethal high temperatures, budding yeast cells can survive for a period of time; however, a sufficient amount of ubiquitin is necessary for this survival. To understand the nature of the stress, we examined the morphological changes in yeast cells, focusing on the vacuoles. Changes in vacuolar morphology were notable, and ruffled vacuolar membranes, accelerated invaginations of vacuolar membranes, and vesicle-like formations were observed. These changes occurred in the absence of Atg1, Atg9 or Ivy1 but appeared to require endosomal sorting proteins, such as Vps23, Vps24 or Pep12. Furthermore, the serial sections of the vacuoles analysed using an electron microscopic analysis revealed that spherical invaginated structures were linked together in a vacuole. Because degradation of cell surface proteins is induced from heat stress, fusion of endosomal and vacuolar membranes might occur frequently in heat-stressed cells, and yeast cells might be able to cope with a rapid increase in vacuolar surface area by such invaginations.