Melatonin protects oogenesis from hypobaric hypoxia-induced fertility damage in mice.

Environmental hypoxia adversely affects reproductive health in humans and animals at high altitudes. Therefore, how to alleviate the follicle development disorder caused by hypoxia exposure and to improve the competence of fertility in plateau non-habituated female animals are important problems to be solved urgently. In this study, a hypobaric hypoxic chamber was used for 4 weeks to simulate hypoxic conditions in female mice, and the effects of hypoxia on follicle development, proliferation and apoptosis of granulosa cells, reactive oxygen species (ROS) levels in MII oocyte and 2-cell rate were evaluated. At the same time, the alleviating effect of melatonin on hypoxic exposure-induced oogenesis damage was evaluated by feeding appropriate amounts of melatonin daily under hypoxia for 4 weeks. The results showed that hypoxia exposure significantly increased the proportion of antral follicles in the ovary, the number of proliferation and apoptosis granulosa cells in the follicle, and the level of ROS in MII oocytes, eventually led to the decline of oocyte quality. However, these defects were alleviated when melatonin was fed under hypoxia conditions. Together, these findings suggest that hypoxia exposure impaired follicular development and reduced oocyte quality, and that melatonin supplementation alleviated the fertility reduction induced by hypoxia exposure.

Zichong granules promote differentiation of ovarian granulosa-like cells from human embryonic stem cells in vitro.

OBJECTIVE: To investigate whether Zichong granules (, ZCKL), a very effective herbal formula for treating infertility, have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells (hESCs) in vitro, and to explore the cellular mechanisms of its clinical effects. METHODS: Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from hESCs for 6 d. Normal rat serum and a set of growth factors were used as negative and positive controls, respectively. RESULTS: ZCKL-medicated rat serum, but not normal rat serum, induced hESCs-derived mesoderm cells to differentiate into functional ovarian granulosa-like cells (OGLCs) in a similar manner to defined growth factors. The induced OGLCs resembled the morphology of native human granulosa cells, expressed granulosa cell-specific markers at both the mRNA and protein levels, produced high levels of estradiol and strongly responded to follicle-stimulating hormone stimulation. Furthermore, mRNA levels of follistatin, mothers against decapentaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process. CONCLUSION: In the ZCKL treatment of infertility, one mechanism by which ZCKL may act is by influencing ovarian granulosa cell differentiation and development, possibly through the follistatin and BMP/SMAD signaling pathways.

Maternal dietary supplementation with grape seed extract in reproductive hens increases fertility in females but decreases semen quality in males of the F1 generation.

Genetic selection in parental broiler breeders has increased their susceptibility to metabolic disorders and reproductive dysfunction. We have recently shown that maternal dietary grape seed extract (GSE) supplementation in hens improves fertility parameters, egg quality, oxidative stress in different tissues and the quality of F1 chicks. Here, we analysed the growth and fertility (both female and male) of the F1 generation animals and the quality of their offspring (F2 generation). Eggs issued from hens supplemented with GSE presented lower ROS production than control hens, suggesting a change in the embryonic environment. However, this did not affect the growth nor the body composition of male and female F1s from hatching to adulthood (37 weeks of age). At 37 weeks of age, the biochemistry analysis of the GSE-F1 muscle has revealed an increase in sensitivity to oxidative stress and a slight change in lipid composition. Both male and female F1-GSE groups presented a delay in puberty with a lower testis volume at 30 weeks of age and lower ovary development at 26 weeks of age. Adult GSE-F1 males did not present histological alterations of seminiferous tubules or semen production, but the semen quality was degraded due to higher oxidative stress and DNA-damaged spermatozoa compared with control F1 animals. In adult GSE-F1 females, despite the delay in puberty, the females laid more eggs of better quality (fewer broken eggs and a higher hatching rate). At hatching, the weight of the chicks from GSE-F1 females was reduced, and this effect was stronger in F2 male chicks (F2) compared with F2 control chicks (F2), because of the lower muscle volume. In conclusion, we can raise the hypothesis that maternal dietary GSE supplementation produces eggs with change in embryonic metabolism, which may affect in adulthood the fertility. The data obtained from the F1-GSE group pointed to a sex-specific modification with higher egg quality in females but semen sensitive to stress in males. Finally, male F2 chicks were leaner than control chicks. Thus, maternal dietary grape seed extract (GSE) supplementation in hens may impact on the fertility of the offspring in a sex-specific manner in subsequent generations.

Bee pollens originating from different species have unique effects on ovarian cell functions.

CONTEXT: The species-specific differences and mechanisms of action of bee pollen on reproduction have not been well studied. OBJECTIVE: We compared the effects of bee pollen extracts from different plants on ovarian cell functions. MATERIALS AND METHODS: We compared the effects of pollens from black alder, dandelion, maize, rapeseed, and willow at 0, 0.01, 0.1, 1, 10, or 100 µg/mL on cultured porcine ovarian granulosa cells. Cell viability was assessed with a Trypan blue test, the cell proliferation marker (PCNA), and an apoptosis marker (BAX) were assessed by immunocytochemistry. Insulin-like growth factor (IGF-I) release was measured by an enzyme-linked immunosorbent assay. RESULTS: Addition of any bee pollen reduced cell viability, promoted accumulation of both proliferation and apoptosis markers, and promoted IGF-I release. The ability of various pollens to suppress cell viability ranked as follows: rapeseed > dandelion > alder > maize > willow. The biological activity of bee pollens regarding their stimulatory action on ovarian cell proliferation ranked as follows: dandelion > willow > maize > alder > rapeseed. Cell apoptosis was promoted by pollens as follows: range > dandelion > alder > rapeseed > willow > maize. The ability of the pollens to stimulate IGF-I output are as follows: willow > dandelion > rapeseed > maize > alder. DISCUSSION: Bee pollen can promote ovarian cell proliferation by promoting IGF-I release, but it induces the dominance of apoptosis over proliferation and the reduction in ovarian cell viability in a species-specific manner. CONCLUSIONS: This is the first demonstration of adverse effects of bee pollen on ovarian cell viability and of its direct stimulatory influence on proliferation, apoptosis, and IGF-I release. The biological potency of bee pollen is dependent on the plant species.

Viburnumfocesides A - D, 1-O-isovaleroylated iridoid 11-O-alloside derivatives from Viburnum foetidum var. ceanothoides.

Viburnumfocesides A - D, four undescribed 1-O-isovaleroylated iridoid 11-O-allosides modified with (Z / E)-p-coumaric acid, were isolated from the aqueous EtOH extract of the twigs of Viburnum foetidum var. ceanothoides, together with seven known natural products. Their structures were identified on the basis of the spectroscopic data interpretation and chemical derivation studies. Cell-based estrogen biosynthesis assays indicated that viburnumfoceside D (4), (2S,3R)-2,3-dihydro-3-hydroxymethyl-7-methoxy-2-(4-hydroxy-3-methoxyphenyl)-5-benzofuranpropanol-3a-O-α-L-rhamnopyranoside (8), and (-)-eriodictyol (11) inhibit estrogen biosynthesis with IC50 values of 5.8, 1.5, and 1.1 µM, respectively, in human ovarian granulosa-like KGN cells via decreasing the expression level of aromatase.

Dendrobium nobile Lindl. polysaccharides improve follicular development in PCOS rats.

Polycystic ovary syndrome (PCOS) is the most typical and common metabolic abnormalities in women of reproductive age. This study examined the protective effects of Dendrobium nobile Lindl. polysaccharides (DNLP) on ovarian follicular development in letrozole-induced PCOS rats and explored the underlying molecular mechanisms. The PCOS rats showed the increased body weight, serum testosterone and luteinizing hormone levels and insulin resistance. DNLP treatment reduced the body weight, serum testosterone level and insulin resistance, but failed to affect luteinizing hormone level in the PCOS rats. DNLP treatment recovered disrupted estrous cycle in the PCOS rats. DNLP treatment decreased antral follicles and increased the thickness of the granular cell layer. DNLP treatment increased the PCNA mRNA and protein expression levels in the PCOS ovarian tissues, and inhibited cell apoptosis in the PCOS ovarian tissues via regulating apoptosis-related proteins including Bax, Bcl-2 and caspase-3. In summary, this study demonstrated the protective effects of DNLP on the ovaries in the letrozole-induced PCOS rat model. DNLP exerted its protective effects via improving follicular development and inhibiting apoptosis of ovarian granular cells in PCOS rats. This study will provide experimental basis for the future clinical application of DNLP in the treatment of PCOS.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development.

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

Roles of different n-3/n-6 PUFA ratios in ovarian cell development and steroidogenesis in PCOS rats.

Polycystic ovary syndrome (PCOS) is a complex and common endocrine disorder characterized by hyperandrogenism, which is accompanied by follicle growth arrest at the small antral stage, minimal granulosa cell proliferation, and chronic anovulation. Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Although PUFAs play an important role in the regulation of female reproduction, their role in ovarian development in PCOS is still unclear. The present study was conducted to investigate the effects of different ratios of n-3/n-6 PUFAs (omega-3/omega-6) on ovary development in PCOS rats. Serum levels of reproductive hormones and enzymes related to steroidogenesis were assessed. The results indicated that PUFAs (n-3/n-6: 1/15) significantly increased ovarian weight and improved the ovarian structure although they had no significant effect on body weight in PCOS rats. Meanwhile, apoptosis was attenuated accompanied by increased cell proliferation by PUFAs (n-3/n-6: 1/15). Moreover, serum levels of hormones (FSH and E2) were also significantly increased by PUFAs (n-3/n-6: 1/15) accompanied by decreased T levels. To investigate whether PUFAs regulate the expression of enzymes related to hormone synthesis, western blotting was used to determine the protein levels of CYP51, CYP19, StAR and 3ß-HSD. The results showed that PUFAs significantly increased the protein levels of all of these enzymes. These results indicate that PUFAs enhance the reproductive performance of PCOS by increasing the expression of steroidogenesis enzymes, which are related to hormone secretion and ovarian functions. These findings provide evidence that a balanced n-3/n-6 PUFA ratio is beneficial for PCOS reproduction.

Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure.

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.

Moxibustion improves ovary function by suppressing apoptosis events and upregulating antioxidant defenses in natural aging ovary.

AIMS: Ovarian aging is a natural physiological phenomenon accompanied by follicular atresia as well as the decline of oocyte quality. Moxibustion is a form of traditional Chinese medicine therapy which has been reported to treat many aging-related problems and improve immune defense. MATERIALS AND METHODS: Moxibustion treatment was applied to the 10-month female rats for 2 or 6 months to evaluate whether moxibustion could delay ovarian aging. The expression levels of NQO-1, HO-1, Bax and Bcl-2 were examined by Western blotting. The serum levels of E2 and FSH concentration were measured through ELISA. P21, P16, NQO-1, HO-1, Bax and Bcl-2 were measured by qRT-PCR. KEY FINDINGS: We demonstrated that moxibustion treatment could attenuate oxidative stress and apoptosis in ovaries, which lead to ovarian aging. The ovary histomorphology, serum FSH, E2 levels as well as aging markers P21 and P16 expression were compared among the groups, which showed that moxibustion treatment could alleviate the ovary fibrosis, decrease the aging markers expression and increase secretion of ovary functional hormones. The mRNA and protein expression levels of the antioxidative stress-related genes HO-1 and NQO-1 were increased after moxibustion treatment. The antiapoptotic factor Bcl-2 and proapoptotic factor Bax were also detected by qRT-PCR and western blotting, and the results demonstrated that moxibustion significantly downregulated the ratio of Bax/Bcl-2, suggesting that moxibustion could reduce apoptosis in the ovaries of aged rats. SIGNIFICANCE: In conclusion, our research revealed that moxibustion could improve ovary function by suppressing apoptosis events and upregulating antioxidant defenses in the natural aging ovary.

Dietary Fish Oil and Vitamin E Reduce Cryoinjuries and Apoptosis in Vitrified Laying Hens' Ovarian Tissue.

The occurrence of cryoinjuries during ovarian tissue freezing necessitates development of methods that can overcome these challenges in cell and tissue cryopreservation. It has been hypothesized that omega-3 and vitamin E diet supplementation to the animal could be an appropriate strategy to preserve post-thaw ovarian quality. The laying hen is an appropriate animal model that can be used to study the effects of providing dietary supplements on the outcomes of ovary cryopreservation. The purpose of the study was to evaluate the dietary effects of fish oil, sunflower oil, and vitamin E on 68 laying hens according to the following treatments: basal diet + 1.5% sunflower oil (control; C); basal diet + 1.5% sunflower oil + 1.1 IU alpha-tocopherol/hen/day (E); basal diet + 1.5% fish oil + 1.1 IU alphatocopherol/hen/day (n-3+E); and basal diet + 1.5% fish oil (n-3). The effects on ovarian structure and preservation, apoptosis-related gene expression, and the fatty acid profiles in ovarian laying hen (n = 7 in each group) were studied. The number of intact primordial follicles in n-3+E group was significantly higher than other groups (85% vs. 71%, 72%, and 77% for n-3+E, C, E, and n-3, respectively; p < 0.01). There was a significant reduction in expression of Cas3, as well as Cas8, in n-3 and n-3+E than C and E (p ≤ 0.05). A trend to decrease in Bak (p = 0.089) and Bak/Bcl2 ratio (p = 0.095) in the mRNA was observed in n-3+E. Ovarian eicosapentaenoic acid (C20:5n3) concentration in n-3 was the highest among C, E, and n-3+E (p < 0.01). Docosahexaenoic acid (C22:6 n-3) concentrations in ovaries of the n-3 group were elevated five times more than control. The n-3: n-6 ratio in groups receiving omega-3 (n-3+E and n-3) was higher than other groups (p < 0.01). In conclusion, consumption of dietary omega-3 fatty acids with vitamin E improves the results of vitrification of ovarian tissues in laying hens.

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

Direct inhibitory effect of flaxseed on porcine ovarian granulosa cell functions.

Flaxseed is useful as a functional food and alternative medicine owing to its beneficial health effects. Its action on ovarian cell functions and interrelationships with the upstream hormonal regulators remain unknown. Our aim was to examine the direct influence of flaxseed extract on basal porcine ovarian functions (proliferation, apoptosis), leptin release, and response to insulin-like growth factor I (IGF-I). First, we examined the effect of flaxseed extract on the accumulation of proliferation (PCNA) and apoptosis (Bax) markers and on leptin release in cultured porcine ovarian granulosa cells. Next, granulosa cells were cultured with IGF-I with and without flaxseed extract and analyzed for PCNA and Bax accumulation by quantitative immunocytochemistry and for leptin release by radioimmunoassay. Flaxseed decreased the accumulation of PCNA and increased that of Bax at all doses and reduced leptin output at 100 µg/mL. In contrast, IGF-I promoted PCNA accumulation and suppressed Bax. Flaxseed did not modify IGF-I action on these parameters. Thus, we showed that flaxseed influences porcine reproductive processes, having a direct effect on the ovary and the ability to affect ovarian cell proliferation, apoptosis, and leptin release. Furthermore, we confirmed the pro-proliferative and antiapoptotic actions of IGF-I but showed that flaxseed action on ovarian cell proliferation and apoptosis is not due to changes in the cell response to IGF-I. The potential direct anti-reproductive action of flaxseed needs to be considered during its application in nutrition, medicine, and animal production.

Low level laser therapy (LLLT) modulates ovarian function in mature female mice.

It is known that LLLT has beneficial effects on several pathological conditions including wound healing, pain and inflammation. LLLT modulates biological processes, including cell proliferation, apoptosis and angiogenesis. In the present study, we examined the effect of local application of LLLT on follicular dynamics, ovarian reserve, AMH expression, progesterone levels, apoptosis, angiogenesis, and reproductive outcome in adult mice. LLLT (200 J/cm2) increased the percentage of primary and preantral follicles, whilst decreasing the percentage of corpora lutea compared to control ovaries. LLLT-treated ovaries did not exhibit any changes regarding the number of primordial follicles. We observed a higher percentage of AMH-positive follicles (in early stages of development) in LLLT-treated ovaries compared to control ovaries. LLLT reduced the P4 concentration and the apoptosis in early antral follicles compared to control ones. LLLT caused a reduction in the endothelial cell area and an increase in the periendothelial cell area in the ovary. Additionally, LLLT was able to improve oocyte quality. Our findings suggest that local application of LLLT modulates follicular dynamics by regulating apoptosis and the vascular stability in mouse ovary. In conclusion, these data indicate that LLLT might become a novel and useful tool in the treatment of several pathologies, including female reproductive disorders.

A unified scale for female reproductive stages in the Norway lobster (Nephrops norvegicus): Evidence from macroscopic and microscopic characterization.

Knowledge of the reproductive cycle in exploited species is important for a sustainable management of fisheries. Standardized scales to assess maturity stages are a fundamental tool to understand the demographic composition of exploited populations. Staging scales for female Norway lobster, Nephrops norvegicus, have been subject to a series of changes, and multiple inconsistent scales are in use in different fisheries regions. A unified, evidence-based scale has not previously been established. We reviewed previous staging scales for the female ovary maturation and propose a revised scale based on the correlation between macroscopic and microscopic ovary characteristics. To provide better-informed tools for future stock assessment, female stages were characterized through external observation on ovary color and size, and the progress of vitellogenesis. This study clarifies several biological phases that were conflated in previous scales. First, we demonstrate how to distinguish between immature ovaries in juvenile females versus the earliest ovary maturation stage in adults. Second, the new scale differentiates between "mottled" ovaries seen in two separate biological stages: the spent ovaries that undergo partial resorption in berried females, versus ovaries of females which failed to spawn and undergo full resorption. To ensure consistent application, colors are assessed relative to international standards (RAL/Pantone). This new, practical staging scheme clarifies the correlation between microscopic characteristics and macroscopically observable details in ovary maturation. Adoption of this unified staging scale will improve maturity analyses, help to identify stocks with potentially reduced reproductive capacity, and facilitate broad-scale comparisons.

Functional anatomy of the female reproductive system of the American lobster (Homarus americanus).

Light microscopy studies of the female American lobster Homarus americanus reproductive system are essentially nonexistent or outdated. Based on samples taken in the spring, summer, and autumn from the southern Gulf of St. Lawrence between 1994 and 2014, and using a combination of histological and scanning electron microscope techniques, we propose an ovarian cycle with 10 stages, identifying for the first time a recovery stage. Also, an atypical resorption stage, characterized by massive reabsorption of mature oocytes, is occasionally observed during summer months. The oviducts are composed of connective tissue (elastic and collagen fibers) with no muscle or secretory activities. Their epithelium shows a cyclic pattern and phagocytosis activities linked to spawning. Although the role of the seminal receptacle is to store and protect semen, free spermatozoa (i.e., without the spermatophoric wall and the acellular gelatinous substance that constitute the semen) were also observed in its posteriolateral grooves immediately prior to spawning, which is consistent with an external fertilization mechanism at the seminal receptacle. Unexpectedly, free spermatozoa were observed externally near two pore-like structures located on the gonopore's operculum, not at the seminal receptacle, after spawning; hence, more work is needed to fully understand the fertilization mechanism for the American lobster.

Morphological alterations in the ovaries of Amblyomma cajennense semi-engorged ticks exposed to ethanolic extract of Acmella oleracea.

The present study analyzed the effects of different concentrations of Acmella oleracea crude ethanolic extract (EEAO) on the development of germ cells from semi-engorged Amblyomma cajennense females in order to evaluate the potential of this natural chemical as a strategy to control these important ectoparasites. A hundred semi-engorged females were divided into five groups (duplicates) (10 animals/group): Control 1 (distilled water); Control 2 (solvent ethanol 50% and DMSO 1%); and Treatment I to III (3.1, 6.2, and 12.5 mg/mL of EEAO, respectively). For the exposure of the ticks to the extract was used the Adult Immersion Test. After the exposition, the ovaries were removed and submitted to histological analysis using Harris hematoxylin and aqueous eosin. The histochemical tests were performed using PAS and Bromophenol blue staining techniques, for the detection of total polysaccharides and total protein, respectively. The extract caused significant alterations in the oocytes, including changes in the shape of the cells, disorganization, and cytoplasmic vacuolation, decrease in the number of yolk granules and germ vesicle fragmentation. These alterations were more intense in the oocytes in initial developmental stages (I and II). The results obtained in this study confirm the cytotoxic potential of the ethanolic extract of A. oleracea on the germ cells of A. cajennense females, opening up the possibility to use this extract as an alternative to control these ectoparasites.

Effect of Catalase or Alpha Lipoic Acid Supplementation in the Vitrification Solution of Ovine Ovarian Tissue.

AIM: The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue. METHODS: This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL-1) or ALA (25, 50, or 100 µM mL-1). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels. RESULTS: The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 µM mL-1 than those observed for the treatments INC, CAT (40 IU mL-1), or ALA (25 or 50 µM mL-1). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL-1), ROS levels (10 and 20 IU mL-1), as well as DNA damage revealed by ©H2AX (40 IU mL-1). CONCLUSIONS: Although 100 µM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.

Supplemental flaxseed modulates ovarian functions of weanling gilts via the action of selected fatty acids.

The aim of this study was to examine the influence of dietary flaxseed on the endocrine and ovarian functions of weanling gilts challenged with E. coli and Coronavirus infections treated with dietary probiotic cheeses and to understand the possible mechanisms of its effects on ovarian function. Probiotics were used as a natural substitution for antibiotics and 10% dietary flaxseed is an effective prebiotic which supports the action of probiotics and has other beneficial effects on the organism. Probiotics with or without flaxseed were fed to weanling gilts starting 10 days before and lasting up until 14 days after weaning. The ovaries were measured and histologically analysed. The blood samples for the levels of steroid hormones and insulin-like growth factor I (IGF-I) were assessed using immunoassays and the levels of fatty acids were assessed using gas chromatography. All samples were collected on the day of weaning and 14 days after weaning. On the day of weaning, increased levels of linoleic acid and IGF-I was associated with higher body weight. The steroid hormones were not affected by the diet. The conversion of alpha-linolenic acid (ALA) to timodonic (EPA) and cervonic (DHA) acids were lower compared to controls, and together with high levels of myristic, palmitic and palmitoleic acids was associated with the higher proliferation and lower apoptosis in the primordial, primary and secondary follicles; although the inhibition of the cell cycle was observed in relation to the low level of eicosadienoic acid. The high levels of ALA, EPA and DHA and the low levels of myristic, palmitic and palmitoleic acids may have been the effect of flaxseed feeding 14 days post-weaning and may have had a reverse effect on the proliferation and apoptosis of ovarian follicles. These data suggest that flaxseed may suppress the follicle development in weanlings via the stimulation of apoptosis and the inhibition of proliferation via the modulation of the metabolism of selected fatty acids.