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Ketoconazole (KTZ) is widely used as a fungicide, but it is also known to target steroid hormone formation which may affect female reproductive health. Our study aims to investigate the effects of KTZ on in vitro matured bovine cumulus-oocyte complexes (COCs), as a model for female reproductive toxicity. Cumulus cells of in vitro maturing COCs produce progesterone and pregnenolone, but exposure to 10 -6M KTZ effectively blocked the synthesis of these hormones. Exposure to lower concentrations of KTZ (i.e. 10 -7M and 10 -8M) had no such effect on steroidogenesis compared to the 0.1% v/v DMSO vehicle control. Classical parameters of in vitro COC maturation, such as oocyte nuclear maturation to the metaphase II stage and expansion of the cumulus investment, were not affected by any KTZ concentration tested. Apoptosis and necrosis levels were also not altered in cumulus cells or oocytes exposed to KTZ. Moreover, oocytes exposed to KTZ during maturation showed normal cleavage and early embryo development up to day 8 post fertilization; albeit a statistically significant decrease was observed in day 8 blastocysts produced from oocytes exposed to the lowest concentration of 10 -8M KTZ. When unexposed mature oocytes were fertilized, followed by embryo culture for 8 days under KTZ exposure, no adverse effects in embryo cleavage and blastocyst formation were observed. In conclusion, KTZ has no major impact on in vitro bovine oocyte maturation and blastocyst formation in our study, even at concentrations blocking steroidogenesis.

Obese women are subfertile and have reduced assisted reproduction success, which may be due to reduced oocyte competence. We hypothesize that consumption of a high-fat/high-sugar diet induces ovarian inflammation, which is a primary contributor to decreased oocyte quality and pre-implantation embryo development. To test this hypothesis, C57BL/6 (B6) mice with a normal inflammatory response and C3H/HeJ (C3H) mice with a dampened inflammatory response due to dysfunctional Toll-like receptor 4 were fed either normal chow or high-fat/high-sugar diet. In both B6 and C3H females, high-fat/high-sugar diet induced excessive adiposity and hyperglycemia compared to normal chow-fed counterparts. Conversely, ovarian CD68 levels and oocyte expression of oxidative stress markers were increased when collected from B6 high-fat/ high-sugar but not C3H high-fat/high-sugar mice. Following in vitro fertilization of in vivo matured oocytes, blastocyst development was decreased in B6-high-fat/high-sugar but not C3H high-fat/high-sugar mice. Expression of cumulus cell markers of oocyte quality were altered in both B6 high-fat/high-sugar and C3H high-fat/high-sugar. However, there were no diet-dependent differences in spindle abnormalities in either B6 or C3H mice, suggesting potential defects in cytoplasmic maturation. Indeed, there were significant increases in the abundance of maternal effect gene mRNAs in oocytes from only B6 high-fat/high-sugar mice. These differentially expressed genes encode proteins of the subcortical maternal complex and associated with mRNA metabolism and epigenetic modifications. These genes regulate maternal mRNA degradation at oocyte maturation, mRNA clearance at the zygotic genome activation, and methylation of imprinted genes suggesting a mechanism by which inflammation induced oxidative stress impairs embryo development.

Objective To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. Design Descriptive study. Setting University Hospital. Patients The study included samples from 121 patients: 71 patients (aged 17–37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20–35 years) receiving in vitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. Interventions None. Main Outcome Measures MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. Results Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian c, Objective: To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. Design: Descriptive study. Setting: University Hospital. Patients: The study included samples from 121 patients: 71 patients (aged 17–37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20–35 years) receiving in vitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. Interventions: None. Main Outcome Measures: MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. Results: Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10 -9 M, 10 -7 M, 10 -5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10 -9 M or 10 -7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10 -5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e

In G. sigillatus, the ovaries are panoistic type in which the ovarioles exhibit a progressive increase in dimension as the development proceeds. The yolk deposition was found to commence on the 4th day of adult emergence in the basal oocyte which attained a fully mature stage on day 8. During this process, considerable changes were observed in both the oocytes and the follicular cells from the second day of female adult emergence. In the earlier adult instars, yolk spheres appeared in the cortical region of the ooplasm and large yolk spheres in the central region during vitellogenic phase. At the time of vitellogenesis, the follicular epithelial cells of the basal oocytes were tall and columnar with intercellular spaces. As the vitellogenesis progressed, the cells became cuboidal and the oocytes developed vitelline membrane and chorion. The present study also reveals that vitellogenesis in Gryllodes sigillatus occurs both in the penultimate and basal oocytes simultaneously.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

The luteinizing hormone (LH) and maturation-inducing steroids (MIS), such as 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, regulate the final oocyte maturation in teleosts. Oocyte maturational competence (OMC) and ovulatory competence measure the sensitivity to MIS for oocyte maturation and ovulation, respectively. However, the molecular mechanisms underlying the acquisition of ovulatory competence remain unknown. Sturgeons are an excellent research model for investigating these mechanisms. We examined the seasonal profiles of OMC and ovulatory competence in vitro and the expression of 17 ovulation-related gene candidates using quantitative PCR in Amur sturgeon ovarian follicles. The ovulatory competence was induced by the LH-releasing hormone analog (LHRHa) priming injection after acquiring the OMC, which was spontaneously induced in spring or autumn. Seven genes, including the tissue-type plasminogen activator (plat), were enhanced following the LHRHa priming injection in ovarian follicles sampled from anovulated and ovulated fish. The activin receptor type 1 (acvr1) and prostaglandin G/H synthase 2 (ptgs2) were only upregulated in ovulated fish. Our results suggest that plat/plasmin and prostaglandin (PG)/PG receptor systems are essential for sturgeon ovulation, similar to other vertebrates. Notably, successful ovulation depends on a sufficient PG synthesis, and mediators activating the PG/PG receptor system are essential for acquiring the ovulatory competence. We provide the first report of ovulation-related gene alterations in the ovarian follicles of Amur sturgeons.

In G. sigillatus, the ovaries are panoistic type in which the ovarioles exhibit a progressive increase in dimension as the development proceeds. The yolk deposition was found to commence on the 4th day of adult emergence in the basal oocyte which attained a fully mature stage on day 8. During this process, considerable changes were observed in both the oocytes and the follicular cells from the second day of female adult emergence. In the earlier adult instars, yolk spheres appeared in the cortical region of the ooplasm and large yolk spheres in the central region during vitellogenic phase. At the time of vitellogenesis, the follicular epithelial cells of the basal oocytes were tall and columnar with intercellular spaces. As the vitellogenesis progressed, the cells became cuboidal and the oocytes developed vitelline membrane and chorion. The present study also reveals that vitellogenesis in Gryllodes sigillatus occurs both in the penultimate and basal oocytes simultaneously.

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

The luteinizing hormone (LH) and maturation-inducing steroids (MIS), such as 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, regulate the final oocyte maturation in teleosts. Oocyte maturational competence (OMC) and ovulatory competence measure the sensitivity to MIS for oocyte maturation and ovulation, respectively. However, the molecular mechanisms underlying the acquisition of ovulatory competence remain unknown. Sturgeons are an excellent research model for investigating these mechanisms. We examined the seasonal profiles of OMC and ovulatory competence in vitro and the expression of 17 ovulation-related gene candidates using quantitative PCR in Amur sturgeon ovarian follicles. The ovulatory competence was induced by the LH-releasing hormone analog (LHRHa) priming injection after acquiring the OMC, which was spontaneously induced in spring or autumn. Seven genes, including the tissue-type plasminogen activator (plat), were enhanced following the LHRHa priming injection in ovarian follicles sampled from anovulated and ovulated fish. The activin receptor type 1 (acvr1) and prostaglandin G/H synthase 2 (ptgs2) were only upregulated in ovulated fish. Our results suggest that plat/plasmin and prostaglandin (PG)/PG receptor systems are essential for sturgeon ovulation, similar to other vertebrates. Notably, successful ovulation depends on a sufficient PG synthesis, and mediators activating the PG/PG receptor system are essential for acquiring the ovulatory competence. We provide the first report of ovulation-related gene alterations in the ovarian follicles of Amur sturgeons.

In G. sigillatus, the ovaries are panoistic type in which the ovarioles exhibit a progressive increase in dimension as the development proceeds. The yolk deposition was found to commence on the 4th day of adult emergence in the basal oocyte which attained a fully mature stage on day 8. During this process, considerable changes were observed in both the oocytes and the follicular cells from the second day of female adult emergence. In the earlier adult instars, yolk spheres appeared in the cortical region of the ooplasm and large yolk spheres in the central region during vitellogenic phase. At the time of vitellogenesis, the follicular epithelial cells of the basal oocytes were tall and columnar with intercellular spaces. As the vitellogenesis progressed, the cells became cuboidal and the oocytes developed vitelline membrane and chorion. The present study also reveals that vitellogenesis in Gryllodes sigillatus occurs both in the penultimate and basal oocytes simultaneously.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

In G. sigillatus, the ovaries are panoistic type in which the ovarioles exhibit a progressive increase in dimension as the development proceeds. The yolk deposition was found to commence on the 4th day of adult emergence in the basal oocyte which attained a fully mature stage on day 8. During this process, considerable changes were observed in both the oocytes and the follicular cells from the second day of female adult emergence. In the earlier adult instars, yolk spheres appeared in the cortical region of the ooplasm and large yolk spheres in the central region during vitellogenic phase. At the time of vitellogenesis, the follicular epithelial cells of the basal oocytes were tall and columnar with intercellular spaces. As the vitellogenesis progressed, the cells became cuboidal and the oocytes developed vitelline membrane and chorion. The present study also reveals that vitellogenesis in Gryllodes sigillatus occurs both in the penultimate and basal oocytes simultaneously.

The luteinizing hormone (LH) and maturation-inducing steroids (MIS), such as 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, regulate the final oocyte maturation in teleosts. Oocyte maturational competence (OMC) and ovulatory competence measure the sensitivity to MIS for oocyte maturation and ovulation, respectively. However, the molecular mechanisms underlying the acquisition of ovulatory competence remain unknown. Sturgeons are an excellent research model for investigating these mechanisms. We examined the seasonal profiles of OMC and ovulatory competence in vitro and the expression of 17 ovulation-related gene candidates using quantitative PCR in Amur sturgeon ovarian follicles. The ovulatory competence was induced by the LH-releasing hormone analog (LHRHa) priming injection after acquiring the OMC, which was spontaneously induced in spring or autumn. Seven genes, including the tissue-type plasminogen activator (plat), were enhanced following the LHRHa priming injection in ovarian follicles sampled from anovulated and ovulated fish. The activin receptor type 1 (acvr1) and prostaglandin G/H synthase 2 (ptgs2) were only upregulated in ovulated fish. Our results suggest that plat/plasmin and prostaglandin (PG)/PG receptor systems are essential for sturgeon ovulation, similar to other vertebrates. Notably, successful ovulation depends on a sufficient PG synthesis, and mediators activating the PG/PG receptor system are essential for acquiring the ovulatory competence. We provide the first report of ovulation-related gene alterations in the ovarian follicles of Amur sturgeons.

Purpose: To study the impact of oocyte diameter and cumulus cell mass on the potential for final maturation of immature human oocytes in vitro. Methods: Immature oocytes (n = 1563) from 75 women undergoing fertility preservation by ovarian tissue cryopreservation (14–41 years) were collected. After preparation of the ovarian cortex for freezing, immature oocytes were collected from the surplus medulla. After collection, IVM was performed according to standard published methods. The mass of cumulus cell surrounding the immature oocyte was grouped according to size. After IVM, each oocyte was photographed, measured, and the diameter was calculated as a mean of two perpendicular measurements. Results: The diameter of the oocytes ranged from 60 to 171 µm with a mean of 115 µm (SD:12.1) and an interquartile range from 107 to 124 µm. The oocyte diameter was positively associated with a higher incidence of MII (p < 0.001). MII oocytes had a significantly larger mean diameter than MI, GV, and degenerated oocytes. The size of the cumulus cell mass was significantly associated with the MII stage (p < 0.001) and larger oocyte diameter (p < 0.001). The results further confirm that the diameter of the fully grown oocyte is reached relatively early in human follicular development and that the factors governing oocyte maturation in vitro are connected to the surrounding cell mass and the oocyte. Conclusion: The diameter of the oocyte is a highly determining factor in the nuclear maturation of the human oocyte during in vitro maturation, and the size of the cumulus cell mass is closely positively associated with a larger diameter.

Purpose: To study the impact of oocyte diameter and cumulus cell mass on the potential for final maturation of immature human oocytes in vitro. Methods: Immature oocytes (n = 1563) from 75 women undergoing fertility preservation by ovarian tissue cryopreservation (14–41 years) were collected. After preparation of the ovarian cortex for freezing, immature oocytes were collected from the surplus medulla. After collection, IVM was performed according to standard published methods. The mass of cumulus cell surrounding the immature oocyte was grouped according to size. After IVM, each oocyte was photographed, measured, and the diameter was calculated as a mean of two perpendicular measurements. Results: The diameter of the oocytes ranged from 60 to 171 µm with a mean of 115 µm (SD:12.1) and an interquartile range from 107 to 124 µm. The oocyte diameter was positively associated with a higher incidence of MII (p < 0.001). MII oocytes had a significantly larger mean diameter than MI, GV, and degenerated oocytes. The size of the cumulus cell mass was significantly associated with the MII stage (p < 0.001) and larger oocyte diameter (p < 0.001). The results further confirm that the diameter of the fully grown oocyte is reached relatively early in human follicular development and that the factors governing oocyte maturation in vitro are connected to the surrounding cell mass and the oocyte. Conclusion: The diameter of the oocyte is a highly determining factor in the nuclear maturation of the human oocyte during in vitro maturation, and the size of the cumulus cell mass is closely positively associated with a larger diameter.

Context Supraphysiological sex steroid levels at the follicular-luteal phase transition are implicated as the primary cause of luteal insufficiency after ovarian stimulation (OS) for in vitro fertilization. Objective We aimed to determine the impact of suppressing estradiol levels during OS of multiple dominant follicles on the unsupported luteal phase and markers of endometrial maturation. Methods At 2 university hospitals, 25 eligible egg donors were randomized to undergo OS using exogenous gonadotropins with or without adjuvant letrozole 5 mg/day. Final oocyte maturation was triggered with a GnRH agonist. No luteal support was provided. The primary outcome was the duration of the luteal phase. Secondary outcomes were luteal phase hormone profiles and the endometrial transcriptomic signature 5 days after oocyte pick up (OPU + 5). Results The median (interquartile range [IQR]) luteal phase duration was 8.0 (6.8-11.5) days compared with 5.0 (5.0-6.8) days in the intervention and control group, respectively (P < 0.001). Estradiol levels were effectively suppressed in the letrozole group with a median of 0.86 (0.23-1.24) nmol/L at OPU compared to 2.82 (1.34-3.44) nmol/L in the control group. Median (IQR) progesterone levels at OPU + 5 were 67.05 (15.67-101.75) nmol/L in the letrozole group vs 2.27 (1.05-10.70) nmol/L in the control group (P < 0.001). In the letrozole group, 75% of participants revealed endometrial transcriptomic signatures interpreted as post-receptive. In the control group, 40% were post-receptive and 50% noninformative. Conclusion Suppressing estradiol levels in the follicular phase with adjuvant letrozole significantly reduces the disruption of the unsupported luteal phase after OS.

Research question: Can a methodology be developed for case selection and whole-exome sequencing (WES) analysis of women who are infertile owing to recurrent oocyte maturation defects (OOMD) and/or preimplantation embryo lethality (PREMBL)? Design: Data were collected from IVF patients attending the Istanbul Memorial Hospital (2015–2021). A statistical methodology to identify infertile endophenotypes (recurrent low oocyte maturation rate, low fertilization rate and preimplantation developmental arrest) was developed using a large IVF dataset (11,221 couples). Twenty-eight infertile women with OOMD/PREMBL were subsequently enrolled for WES on their genomic DNA. Pathogenic variants were prioritized using a custom-made bioinformatic pipeline set to minimize false-positive discoveries through resampling in control cohorts (the Human Genome Diversity Project and 1343 whole-exome sequences from oocyte donors). Individual single-cell RNA sequencing data from 18 human metaphase II (MII) oocytes and antral granulosa cells was used for genome-wide validation. WES and bioinformatics were performed at Igenomix and the National Research Council, Italy. Results: Variant prioritization analysis identified 265 unique variants in 248 genes (average 22.4 per sample). Of the genes harbouring high-impact variants 78% were expressed by MII oocytes and/or antral granulosa cells, significantly higher than for random sample of controls (odds ratio = 5, Fisher's exact P = 0.0004). Seven of the 28 women (25%) were homozygous carriers of missense pathogenic variants in known candidate genes for OOMD/PREMBL, including PATL2, NLRP5 (n = 2),TLE6, PADI6, TUBB8 and TRIP13. Furthermore, novel gene–disease associations were identified. In fact, one woman with a low oocyte maturation rate was a homozygous carrier of high-impact variants in ENSA, an essential gene for prophase I meiotic transition in mice. Conclusions: This analytical framework could reveal known and new genes associated with isolat

Research question: Can a methodology be developed for case selection and whole-exome sequencing (WES) analysis of women who are infertile owing to recurrent oocyte maturation defects (OOMD) and/or preimplantation embryo lethality (PREMBL)? Design: Data were collected from IVF patients attending the Istanbul Memorial Hospital (2015–2021). A statistical methodology to identify infertile endophenotypes (recurrent low oocyte maturation rate, low fertilization rate and preimplantation developmental arrest) was developed using a large IVF dataset (11,221 couples). Twenty-eight infertile women with OOMD/PREMBL were subsequently enrolled for WES on their genomic DNA. Pathogenic variants were prioritized using a custom-made bioinformatic pipeline set to minimize false-positive discoveries through resampling in control cohorts (the Human Genome Diversity Project and 1343 whole-exome sequences from oocyte donors). Individual single-cell RNA sequencing data from 18 human metaphase II (MII) oocytes and antral granulosa cells was used for genome-wide validation. WES and bioinformatics were performed at Igenomix and the National Research Council, Italy. Results: Variant prioritization analysis identified 265 unique variants in 248 genes (average 22.4 per sample). Of the genes harbouring high-impact variants 78% were expressed by MII oocytes and/or antral granulosa cells, significantly higher than for random sample of controls (odds ratio = 5, Fisher's exact P = 0.0004). Seven of the 28 women (25%) were homozygous carriers of missense pathogenic variants in known candidate genes for OOMD/PREMBL, including PATL2, NLRP5 (n = 2),TLE6, PADI6, TUBB8 and TRIP13. Furthermore, novel gene–disease associations were identified. In fact, one woman with a low oocyte maturation rate was a homozygous carrier of high-impact variants in ENSA, an essential gene for prophase I meiotic transition in mice. Conclusions: This analytical framework could reveal known and new genes associated with isolat

Ovarian stimulation for oocyte and embryo cryopreservation is the standard of care for fertility preservation in young breast cancer patients before gonadotoxic chemotherapy. The procedure should be started as soon as possible to avoid delay of treatment; thus, it is often performed concomitantly with tumor staging assessments. However, questions remain regarding the potential negative impact on oocyte quality that may occur due to exposure to scattered ionizing radiation from imaging techniques when staging assessment is conducted at the same time as ovarian stimulation., SCOPUS: ar.j, info:eu-repo/semantics/published

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage.

In G. sigillatus, the ovaries are panoistic type in which the ovarioles exhibit a progressive increase in dimension as the development proceeds. The yolk deposition was found to commence on the 4th day of adult emergence in the basal oocyte which attained a fully mature stage on day 8. During this process, considerable changes were observed in both the oocytes and the follicular cells from the second day of female adult emergence. In the earlier adult instars, yolk spheres appeared in the cortical region of the ooplasm and large yolk spheres in the central region during vitellogenic phase. At the time of vitellogenesis, the follicular epithelial cells of the basal oocytes were tall and columnar with intercellular spaces. As the vitellogenesis progressed, the cells became cuboidal and the oocytes developed vitelline membrane and chorion. The present study also reveals that vitellogenesis in Gryllodes sigillatus occurs both in the penultimate and basal oocytes simultaneously.

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF+5% FCS (Control) or supplemented with 5, 10 or 25 mu M nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (C-DMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJ(EGA)) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 +/- 1.0%) and Nob10 (44.4 +/- 1.3%) for MNEGA phase and in Nob10 (61.0 +/- 0.8%) for MJ(EGA) phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPAR alpha transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway

Purpose: Oocyte quality and reproductive outcome are negatively affected by advanced maternal age, ovarian stimulation and method of oocyte maturation during assisted reproduction; however, the mechanisms responsible for these associations are not fully understood. The aim of this study was to compare the effects of ageing, ovarian stimulation and in-vitro maturation on the relative levels of transcript abundance of genes associated with DNA repair during the transition of germinal vesicle (GV) to metaphase II (MII) stages of oocyte development. Method(s): The relative levels of transcript abundance of 90 DNA repair-associated genes was compared in GV-stage and MII-stage oocytes from unstimulated and hormone-stimulated ovaries from young (5-8-week-old) and old (42-45-week-old) C57BL6 mice. Ovarian stimulation was conducted using pregnant mare serum gonadotropin (PMSG) or anti-inhibin serum (AIS). DNA damage response was quantified by immunolabeling of the phosphorylated histone variant H2AX (gammaH2AX). Result(s): The relative transcript abundance in DNA repair genes was significantly lower in MII oocytes compared to GV oocytes in young unstimulated and PMSG stimulated but was higher in AIS-stimulated mice. Interestingly, an increase in the relative level of transcript abundance of DNA repair genes was observed in MII oocytes from older mice in unstimulated, PMSG-stimulated and AIS-stimulated mice. Decreased gammaH2AX levels were found in both GV oocytes (82.9%) and MII oocytes (37.5%) during ageing in both ovarian stimulation types used (PMSG/AIS; p < 0.05). Conclusion(s): In conclusion, DNA repair relative levels of transcript abundance are altered by maternal age and the method of ovarian stimulation during the GV-MII transition in oocytes.Copyright © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

Purpose: Oocyte quality and reproductive outcome are negatively affected by advanced maternal age, ovarian stimulation and method of oocyte maturation during assisted reproduction; however, the mechanisms responsible for these associations are not fully understood. The aim of this study was to compare the effects of ageing, ovarian stimulation and in-vitro maturation on the relative levels of transcript abundance of genes associated with DNA repair during the transition of germinal vesicle (GV) to metaphase II (MII) stages of oocyte development. Method(s): The relative levels of transcript abundance of 90 DNA repair-associated genes was compared in GV-stage and MII-stage oocytes from unstimulated and hormone-stimulated ovaries from young (5-8-week-old) and old (42-45-week-old) C57BL6 mice. Ovarian stimulation was conducted using pregnant mare serum gonadotropin (PMSG) or anti-inhibin serum (AIS). DNA damage response was quantified by immunolabeling of the phosphorylated histone variant H2AX (gammaH2AX). Result(s): The relative transcript abundance in DNA repair genes was significantly lower in MII oocytes compared to GV oocytes in young unstimulated and PMSG stimulated but was higher in AIS-stimulated mice. Interestingly, an increase in the relative level of transcript abundance of DNA repair genes was observed in MII oocytes from older mice in unstimulated, PMSG-stimulated and AIS-stimulated mice. Decreased gammaH2AX levels were found in both GV oocytes (82.9%) and MII oocytes (37.5%) during ageing in both ovarian stimulation types used (PMSG/AIS; p < 0.05). Conclusion(s): In conclusion, DNA repair relative levels of transcript abundance are altered by maternal age and the method of ovarian stimulation during the GV-MII transition in oocytes.Copyright © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF+5% FCS (Control) or supplemented with 5, 10 or 25 mu M nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (C-DMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJ(EGA)) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 +/- 1.0%) and Nob10 (44.4 +/- 1.3%) for MNEGA phase and in Nob10 (61.0 +/- 0.8%) for MJ(EGA) phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPAR alpha transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g(-1) (PFOS-02) representing average human exposure and 53 ng g(-1) (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic ch

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g(-1) (PFOS-02) representing average human exposure and 53 ng g(-1) (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic ch

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g(-1) (PFOS-02) representing average human exposure and 53 ng g(-1) (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic ch

The hormone leptin is a peripheral metabolic signal and an important regulator of energy balance. In mammals, leptin acts on the appetite centers in the hypothalamus, causing anorexigenic functions by inhibiting food intake. It is also considered as a link between the nutritional status and the endocrine reproductive axis. However, the actions of leptin in teleosts are not fully understood. This thesis investigated the possible role of leptin in the regulation of appetite and reproduction in teleosts, using a loss of function leptin receptor zebrafish strain (lepr sa12953). Under different feeding conditions (normal feeding, 7-day fasting, 2- and 6-hours post refeeding) the transcription of orexigenic and anorexigenic genes was influenced by leptin in the zebrafish brain. Leptin signaling inhibited the transcription of orexigenic genes, during short-term fasting and refeeding, and stimulated the transcription of anorexigenic genes under normal feeding in wild-types, indicating an anorexigenic role of leptin in appetite regulation in zebrafish. Moreover, a leptin-dependent gene regulatory network (GRN), involved in the behavioral and metabolic control of appetite was suggested in the brain, including the cart/crhb/gnrh2 genes and their respective co-expressed modules, mediated by the transcription factor sp3a. Furthermore, impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutant females, resulting in low number of ovulated eggs. Moreover, the transcripts of luteinizing hormone beta (lhb) in the pituitary were significantly lower in the mutant females. Analysis of candidate genes revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the ovaries of the lepr mutants. Transcriptomic analysis of isolated fully grown follicles linked the reproductive deficiencies to the suppression of ess

The hormone leptin is a peripheral metabolic signal and an important regulator of energy balance. In mammals, leptin acts on the appetite centers in the hypothalamus, causing anorexigenic functions by inhibiting food intake. It is also considered as a link between the nutritional status and the endocrine reproductive axis. However, the actions of leptin in teleosts are not fully understood. This thesis investigated the possible role of leptin in the regulation of appetite and reproduction in teleosts, using a loss of function leptin receptor zebrafish strain (lepr sa12953). Under different feeding conditions (normal feeding, 7-day fasting, 2- and 6-hours post refeeding) the transcription of orexigenic and anorexigenic genes was influenced by leptin in the zebrafish brain. Leptin signaling inhibited the transcription of orexigenic genes, during short-term fasting and refeeding, and stimulated the transcription of anorexigenic genes under normal feeding in wild-types, indicating an anorexigenic role of leptin in appetite regulation in zebrafish. Moreover, a leptin-dependent gene regulatory network (GRN), involved in the behavioral and metabolic control of appetite was suggested in the brain, including the cart/crhb/gnrh2 genes and their respective co-expressed modules, mediated by the transcription factor sp3a. Furthermore, impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutant females, resulting in low number of ovulated eggs. Moreover, the transcripts of luteinizing hormone beta (lhb) in the pituitary were significantly lower in the mutant females. Analysis of candidate genes revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the ovaries of the lepr mutants. Transcriptomic analysis of isolated fully grown follicles linked the reproductive deficiencies to the suppression of ess

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g(-1) (PFOS-02) representing average human exposure and 53 ng g(-1) (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic ch

The hormone leptin is a peripheral metabolic signal and an important regulator of energy balance. In mammals, leptin acts on the appetite centers in the hypothalamus, causing anorexigenic functions by inhibiting food intake. It is also considered as a link between the nutritional status and the endocrine reproductive axis. However, the actions of leptin in teleosts are not fully understood. This thesis investigated the possible role of leptin in the regulation of appetite and reproduction in teleosts, using a loss of function leptin receptor zebrafish strain (lepr sa12953). Under different feeding conditions (normal feeding, 7-day fasting, 2- and 6-hours post refeeding) the transcription of orexigenic and anorexigenic genes was influenced by leptin in the zebrafish brain. Leptin signaling inhibited the transcription of orexigenic genes, during short-term fasting and refeeding, and stimulated the transcription of anorexigenic genes under normal feeding in wild-types, indicating an anorexigenic role of leptin in appetite regulation in zebrafish. Moreover, a leptin-dependent gene regulatory network (GRN), involved in the behavioral and metabolic control of appetite was suggested in the brain, including the cart/crhb/gnrh2 genes and their respective co-expressed modules, mediated by the transcription factor sp3a. Furthermore, impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutant females, resulting in low number of ovulated eggs. Moreover, the transcripts of luteinizing hormone beta (lhb) in the pituitary were significantly lower in the mutant females. Analysis of candidate genes revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the ovaries of the lepr mutants. Transcriptomic analysis of isolated fully grown follicles linked the reproductive deficiencies to the suppression of ess

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF+5% FCS (Control) or supplemented with 5, 10 or 25 mu M nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (C-DMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJ(EGA)) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 +/- 1.0%) and Nob10 (44.4 +/- 1.3%) for MNEGA phase and in Nob10 (61.0 +/- 0.8%) for MJ(EGA) phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPAR alpha transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway

The hormone leptin is a peripheral metabolic signal and an important regulator of energy balance. In mammals, leptin acts on the appetite centers in the hypothalamus, causing anorexigenic functions by inhibiting food intake. It is also considered as a link between the nutritional status and the endocrine reproductive axis. However, the actions of leptin in teleosts are not fully understood. This thesis investigated the possible role of leptin in the regulation of appetite and reproduction in teleosts, using a loss of function leptin receptor zebrafish strain (lepr sa12953). Under different feeding conditions (normal feeding, 7-day fasting, 2- and 6-hours post refeeding) the transcription of orexigenic and anorexigenic genes was influenced by leptin in the zebrafish brain. Leptin signaling inhibited the transcription of orexigenic genes, during short-term fasting and refeeding, and stimulated the transcription of anorexigenic genes under normal feeding in wild-types, indicating an anorexigenic role of leptin in appetite regulation in zebrafish. Moreover, a leptin-dependent gene regulatory network (GRN), involved in the behavioral and metabolic control of appetite was suggested in the brain, including the cart/crhb/gnrh2 genes and their respective co-expressed modules, mediated by the transcription factor sp3a. Furthermore, impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutant females, resulting in low number of ovulated eggs. Moreover, the transcripts of luteinizing hormone beta (lhb) in the pituitary were significantly lower in the mutant females. Analysis of candidate genes revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the ovaries of the lepr mutants. Transcriptomic analysis of isolated fully grown follicles linked the reproductive deficiencies to the suppression of ess

The hormone leptin is a peripheral metabolic signal and an important regulator of energy balance. In mammals, leptin acts on the appetite centers in the hypothalamus, causing anorexigenic functions by inhibiting food intake. It is also considered as a link between the nutritional status and the endocrine reproductive axis. However, the actions of leptin in teleosts are not fully understood. This thesis investigated the possible role of leptin in the regulation of appetite and reproduction in teleosts, using a loss of function leptin receptor zebrafish strain (lepr sa12953). Under different feeding conditions (normal feeding, 7-day fasting, 2- and 6-hours post refeeding) the transcription of orexigenic and anorexigenic genes was influenced by leptin in the zebrafish brain. Leptin signaling inhibited the transcription of orexigenic genes, during short-term fasting and refeeding, and stimulated the transcription of anorexigenic genes under normal feeding in wild-types, indicating an anorexigenic role of leptin in appetite regulation in zebrafish. Moreover, a leptin-dependent gene regulatory network (GRN), involved in the behavioral and metabolic control of appetite was suggested in the brain, including the cart/crhb/gnrh2 genes and their respective co-expressed modules, mediated by the transcription factor sp3a. Furthermore, impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutant females, resulting in low number of ovulated eggs. Moreover, the transcripts of luteinizing hormone beta (lhb) in the pituitary were significantly lower in the mutant females. Analysis of candidate genes revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the ovaries of the lepr mutants. Transcriptomic analysis of isolated fully grown follicles linked the reproductive deficiencies to the suppression of ess

Perfluorooctane sulfonate (PFOS) has been added to Stockholm Convention for global phase out, but will continue to contribute to the chemical burden in humans for a long time to come due to extreme persistence in the environment. In the body, PFOS is transferred into to the ovarian follicular fluid that surrounds the maturing oocyte. In the present study, bovine cumulus oocyte complexes were exposed to PFOS during 22 h in vitro maturation. Concentrations of 2 ng g(-1) (PFOS-02) representing average human exposure and 53 ng g(-1) (PFOS-53) relevant to highly exposed groups were used. After exposure, developmental competence was recorded until day 8 after fertilisation. Blastocysts were fixed and either stained to evaluate blastomere number and lipid distribution using confocal microscopy or frozen and pooled for microarray-based gene expression and DNA methylation analyses. PFOS-53 delayed the first cleavage to two-cell stage and beyond at 44 h after fertilisation (p < .01). No reduction of proportion blastocysts were seen at day 8 in either of the groups, but PFOS-53 exposure resulted in delayed development into more advanced stages of blastocysts seen as both reduced developmental stage (p = .001) and reduced number of blastomeres (p = .04). Blastocysts showed an altered lipid distribution that was more pronounced after exposure to PFOS-53 (increased total lipid volume, p=.0003, lipid volume/cell p < .0001) than PFOS-02, where only decreased average lipid droplet size (p=.02) was observed. Gene expression analyses revealed pathways differently regulated in the PFOS-treated groups compared to the controls, which were related to cell death and survival through e.g., P38 mitogen-activated protein kinases and signal transducer and activator of transcription 3, which in turn activates tumour protein 53 (TP53). Transcriptomic changes were also associated with metabolic stress response, differentiation and proliferation, which could help to explain the phenotypic ch

Background: Oocyte quality and maturation are influenced by protein supplementation. Objective: To evaluate the influence of fetal bovine serum (FBS) and bovine serum albumin (BSA) concentrations on the recovery and in vitro maturation (IVM) of bovine oocytes. Methods: The study was divided into Stage 1 (oocyte recovery), and Stage 2 (IVM). In the first stage, three experiments were conducted according to the recovery (R) medium used: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; and (R3) the best results from R1, R2, and the combination of FBS+BSA (5+5%). Within the second stage, the maturation medium was supplemented according to three experiments: (M1) 5 vs. 10% FBS; (M2) 0.4 vs. 0.8% BSA; and (M3) better results of M1, M2, and the combination of FBS+BSA (5+0.8%). Results: In Stage 1 (R1 and R2), the media with 10% FBS and 10% BSA showed better oocyte quality results and were defined for experiment R3. In R3, the 10% FBS and the combination of FBS+BSA (5+5%) allowed recovery of better-quality oocytes. In Stage 2 (M1 and M2), media with both levels of FBS (5 and 10%) and 0.8% BSA were defined as better according to the maturation and viability rates of cumulus cells, so they were defined for experiment M3. In M3, no difference was noted among the supplements. Conclusions: For oocyte recovery, 10% FBS and the combination of FBS+BSA (5+5%) can be used to obtain immature oocytes. For the in vitro maturation, FBS (both levels, 5 and 10%) and BSA (0.8%) can be used alone or in combination., Antecedentes: A qualidade e a maturação de oócitos são influenciadas pela suplementação proteica. Objetivo: Avaliar a influência de concentrações de soro fetal bovino (FBS) e albumina sérica bovina (BSA) sobre a recuperação e maturação in vitro (IVM) de oócitos bovinos. Métodos: O estudo foi dividido em Etapa 1 (recuperação de oócitos) e Etapa 2 (IVM). Na primeira etapa, três experimentos foram realizados de acordo com o meio de recuperação: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; e (R3) melhores resultados de R1, R2 e a combinação de FBS+BSA (5+5%). Na segunda etapa, o meio de maturação foi suplementado de acordo com três experimentos: (M1) 5 vs. 10% FBS; (M2) 0,4 vs. 0,8% BSA; e (M3) melhores resultados de M1, M2 e a combinação de FBS+BSA (5+0,8%). Resultados: Na Etapa 1 (R1 e R2), os meios com 10% FBS e 10% BSA mostraram melhores resultados de qualidade oocitária e foram definidos para o experimento R3. Em R3, 10% FBS e a combinação de FBS+BSA (5+5%) permitiram a recuperação de oócitos de melhor qualidade. Na segunda etapa (M1 e M2), meios com ambos os níveis de FBS (5 e 10%) e 0,8% BSA foram definidos como os melhores de acordo com as taxas de maturação e viabilidade de células do cumulus, então foram definidos para o experimento M3. No M3, não houve diferença entre os suplementos. Conclusões: Para a recuperação oocitária, 10% FBS e a combinação de FBS+BSA (5+5%) podem ser usados para obter oócitos imaturos. Para maturação in vitro, FBS (ambos os níveis, 5 e 10%) e BSA (0,8%) podem ser usados sozinhos ou em combinação., Antecedentes: La calidad y maduración de los ovocitos son influenciados por la suplementación proteica. Objetivo: Evaluar la influencia de concentraciones de suero fetal bovino (FBS) y albúmina sérica bovina (BSA) en la recuperación y maduración in vitro (IVM) de ovocitos bovinos. Métodos: El estudio se dividió en Etapa 1 (recuperación de ovocitos) y Etapa 2 (IVM). En la primera etapa, tres experimentos se realizaron de acuerdo con el medio de recuperación: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; y (R3) los mejores resultados de R1, R2 y la combinación de FBS+BSA (5+5%). En la segunda etapa, el medio de maduración fue suplementado para tres experimentos: (M1) 5 vs. 10% FBS; (M2) 0,4 vs. 0,8% BSA; y (M3) mejores resultados de M1, M2 y la combinación de FBS+BSA (5+0,8%). Resultados: En la Etapa 1 (R1 y R2), los medios con 10% FBS y 10% BSA mostraron mejores resultados de calidad oocitaria y fueron definidos para el experimento R3. En R3, 10% FBS y la combinación de FBS+BSA (5+5%) permitieron la recuperación de ovocitos de mejor calidad. En la Etapa 2 (M1 y M2), los medios con ambos niveles de FBS (5 y 10%) y 0,8% de BSA se definieron como mejores de acuerdo con las tasas de maduración y viabilidad de las células del cumulus, por lo que se definieron para el experimento M3. En M3, no se observó diferencia entre los suplementos. Conclusiones: Para la recuperación de ovocitos, se puede utilizar 10% de FBS y la combinación de FBS+BSA (5+5%) para obtener ovocitos inmaduros. Para la maduración in vitro, FBS (ambos niveles, 5 y 10%) y BSA (0,8%) se pueden usar solos o en combinación.

17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20 beta-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17 beta-hydroxysteroid dehydrogenase, type 12-like (17 beta-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17 beta-HSD12 (presumably orthologous to salmon 17 beta-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17 alpha-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20 beta-HSD or 17 beta-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 170HP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17 beta-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17 beta. CR/20 beta-HSD showed strong 17 beta-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 mu g/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 2013-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of

17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20 beta-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17 beta-hydroxysteroid dehydrogenase, type 12-like (17 beta-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17 beta-HSD12 (presumably orthologous to salmon 17 beta-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17 alpha-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20 beta-HSD or 17 beta-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 170HP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17 beta-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17 beta. CR/20 beta-HSD showed strong 17 beta-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 mu g/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 2013-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of

17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20 beta-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17 beta-hydroxysteroid dehydrogenase, type 12-like (17 beta-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17 beta-HSD12 (presumably orthologous to salmon 17 beta-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17 alpha-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20 beta-HSD or 17 beta-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 170HP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17 beta-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17 beta. CR/20 beta-HSD showed strong 17 beta-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 mu g/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 2013-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of

Background: Oocyte quality and maturation are influenced by protein supplementation. Objective: To evaluate the influence of fetal bovine serum (FBS) and bovine serum albumin (BSA) concentrations on the recovery and in vitro maturation (IVM) of bovine oocytes. Methods: The study was divided into Stage 1 (oocyte recovery), and Stage 2 (IVM). In the first stage, three experiments were conducted according to the recovery (R) medium used: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; and (R3) the best results from R1, R2, and the combination of FBS+BSA (5+5%). Within the second stage, the maturation medium was supplemented according to three experiments: (M1) 5 vs. 10% FBS; (M2) 0.4 vs. 0.8% BSA; and (M3) better results of M1, M2, and the combination of FBS+BSA (5+0.8%). Results: In Stage 1 (R1 and R2), the media with 10% FBS and 10% BSA showed better oocyte quality results and were defined for experiment R3. In R3, the 10% FBS and the combination of FBS+BSA (5+5%) allowed recovery of better-quality oocytes. In Stage 2 (M1 and M2), media with both levels of FBS (5 and 10%) and 0.8% BSA were defined as better according to the maturation and viability rates of cumulus cells, so they were defined for experiment M3. In M3, no difference was noted among the supplements. Conclusions: For oocyte recovery, 10% FBS and the combination of FBS+BSA (5+5%) can be used to obtain immature oocytes. For the in vitro maturation, FBS (both levels, 5 and 10%) and BSA (0.8%) can be used alone or in combination., Antecedentes: A qualidade e a maturação de oócitos são influenciadas pela suplementação proteica. Objetivo: Avaliar a influência de concentrações de soro fetal bovino (FBS) e albumina sérica bovina (BSA) sobre a recuperação e maturação in vitro (IVM) de oócitos bovinos. Métodos: O estudo foi dividido em Etapa 1 (recuperação de oócitos) e Etapa 2 (IVM). Na primeira etapa, três experimentos foram realizados de acordo com o meio de recuperação: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; e (R3) melhores resultados de R1, R2 e a combinação de FBS+BSA (5+5%). Na segunda etapa, o meio de maturação foi suplementado de acordo com três experimentos: (M1) 5 vs. 10% FBS; (M2) 0,4 vs. 0,8% BSA; e (M3) melhores resultados de M1, M2 e a combinação de FBS+BSA (5+0,8%). Resultados: Na Etapa 1 (R1 e R2), os meios com 10% FBS e 10% BSA mostraram melhores resultados de qualidade oocitária e foram definidos para o experimento R3. Em R3, 10% FBS e a combinação de FBS+BSA (5+5%) permitiram a recuperação de oócitos de melhor qualidade. Na segunda etapa (M1 e M2), meios com ambos os níveis de FBS (5 e 10%) e 0,8% BSA foram definidos como os melhores de acordo com as taxas de maturação e viabilidade de células do cumulus, então foram definidos para o experimento M3. No M3, não houve diferença entre os suplementos. Conclusões: Para a recuperação oocitária, 10% FBS e a combinação de FBS+BSA (5+5%) podem ser usados para obter oócitos imaturos. Para maturação in vitro, FBS (ambos os níveis, 5 e 10%) e BSA (0,8%) podem ser usados sozinhos ou em combinação., Antecedentes: La calidad y maduración de los ovocitos son influenciados por la suplementación proteica. Objetivo: Evaluar la influencia de concentraciones de suero fetal bovino (FBS) y albúmina sérica bovina (BSA) en la recuperación y maduración in vitro (IVM) de ovocitos bovinos. Métodos: El estudio se dividió en Etapa 1 (recuperación de ovocitos) y Etapa 2 (IVM). En la primera etapa, tres experimentos se realizaron de acuerdo con el medio de recuperación: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; y (R3) los mejores resultados de R1, R2 y la combinación de FBS+BSA (5+5%). En la segunda etapa, el medio de maduración fue suplementado para tres experimentos: (M1) 5 vs. 10% FBS; (M2) 0,4 vs. 0,8% BSA; y (M3) mejores resultados de M1, M2 y la combinación de FBS+BSA (5+0,8%). Resultados: En la Etapa 1 (R1 y R2), los medios con 10% FBS y 10% BSA mostraron mejores resultados de calidad oocitaria y fueron definidos para el experimento R3. En R3, 10% FBS y la combinación de FBS+BSA (5+5%) permitieron la recuperación de ovocitos de mejor calidad. En la Etapa 2 (M1 y M2), los medios con ambos niveles de FBS (5 y 10%) y 0,8% de BSA se definieron como mejores de acuerdo con las tasas de maduración y viabilidad de las células del cumulus, por lo que se definieron para el experimento M3. En M3, no se observó diferencia entre los suplementos. Conclusiones: Para la recuperación de ovocitos, se puede utilizar 10% de FBS y la combinación de FBS+BSA (5+5%) para obtener ovocitos inmaduros. Para la maduración in vitro, FBS (ambos niveles, 5 y 10%) y BSA (0,8%) se pueden usar solos o en combinación.

STUDY QUESTION: How does the human granulosa cell (GC) transcriptome change during ovulation? SUMMARY ANSWER: Two transcriptional peaks were observed at 12 h and at 36 h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. WHAT IS KNOWN ALREADY: The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. STUDY DESIGN, SIZE, DURATION: This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12 h, 17 h or 32 h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36 h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12 h that resembled the initiation of an inflammatory response, and one peak at 36 h that resembled the effector functions of inflammatio