Bovine Ovaries Research Articles

Wingless-type mouse mammary tumor virus integration site regulation of bovine theca cells.

Ovarian paracrine mediation by components of the wingless-type mouse mammary tumor virus integration site ligands (WNT1 to 11) and their receptors, frizzled family members (FZD1 to 10), has been proposed. Secreted truncated forms of FZD proteins (e.g., secreted frizzled-related protein 4 [SFRP4]) block the action of WNT ligands. Dickkopf-1 (DKK1) is another WNT antagonist, and R-spondin-1 (RSPO1) is one of a group of four secreted proteins that enhance WNT/β-catenin signaling. Our hypothesis was that granulosa cells signal theca cells (TCs) via SFRP4, DKK1, RSPO1, and WNT secretion to regulate TC differentiation and proliferation. Therefore, in vitro experiments were conducted to study the effects of WNT family member 3A (WNT3A), WNT5A, RSPO1, DKK1, insulin-like growth factor 1 (IGF1), bone morphogenetic protein 7 (BMP7), Indian hedgehog (IHH), and fibroblast growth factor 9 (FGF9) on bovine TC proliferation and steroidogenesis. TCs of large (8 to 20 mm) and small (3 to 6 mm) follicles were collected from bovine ovaries; TC monolayers were established in vitro and treated with various doses of recombinant human WNT3A, WNT5A, RSPO1, DKK1, IGF1, FGF9, BMP7, IHH, and/or ovine luteinizing hormone (LH) in serum-free medium for 48 h. In experiment 1, using LH-treated TC, IGF1, IHH, and WNT3A increased (P < 0.05) cell numbers and androstenedione production, whereas WNT3A and BMP7 inhibited (P < 0.05) progesterone production. In experiment 2, FGF9 blocked (P < 0.05) the WNT3A-induced increase in androstenedione production in LH plus IGF1-treated TC. In experiment 3, RSPO1 further increased (P < 0.05) LH plus IGF1-induced progesterone and androstenedione production. In experiment 4, SFRP4 and DKK1 alone had no significant effect on TC proliferation or progesterone production of large-follicle TC but both blocked the inhibitory effect of WNT5A on androstenedione production. In contrast, DKK1 alone inhibited (P < 0.05) small-follicle TC androstenedione production whereas SFRP4 was without effect. We conclude that the ovarian TC WNT system is functional in cattle, with WNT3A increasing proliferation and androstenedione production of TC.

The potential of oocytes based on the status of ovary activity to achieve the level of in vitro maturation in Bali cattle

The physiological condition of slaughterhouse bovine ovaries may vary greatly, including the luteal phase, follicular phase, and pre-puberty. However detailed information is scarce on how it affects oocyte maturity in vitro. Therefore, this work aimed to investigate the impacts of ovarian activities on the oocyte maturity of Bali cattle in vitro. Completely randomized designed was arranged, consisting of three ovarian stages (luteal, follicular, and pre-cycle) as a research variable and carried out at four replication. Oocytes were recovered by slicing the follicles from surface-visible ovaries. The oocyte maturation was performed in an incubator containing 5% CO2 at 38.5°C. Following the removal of the cumulus, the oocyte was prepared for fixation, then observed under a microscope. The research showed that the structure of oocyte grades A (compact cumulus cells and thick cytoplasm) in the follicular and luteal phase was similar, but such condition was significantly different compared to the prepubertal phase. The oocyte maturity of M-II was significantly lower (P<0.05) in the pre-cycle phase than in the luteal and follicular phase. Meanwhile, the stages of GV, GVBD, and MI appeared to be identical in all phases. This work concluded that the structure of the oocyte population is closely related to ovarian stages, resulting in higher maturity on M-II oocytes during the luteal and follicular phases.

Similarities between bovine and human germline development revealed by single-cell RNA sequencing.

The germ cell lineage ensures the creation of new individuals and perpetuates the genetic information across generations. Primordial germ cells are pioneers of gametes and exist transiently during development until they differentiate into oogonia in females, or spermatogonia in males. Little is known about the molecular characteristics of primordial germ cells in cattle. By performing single-cell RNA-sequencing, quantitative real-time PCR, and immunofluorescence analyses of fetal gonads between 40 and 90 days of fetal age, we evaluated the molecular signatures of bovine germ cells at the initial stages of gonadal development. Our results indicate that at 50 days of fetal age, bovine primordial germ cells were in the early stages of development, expressing genes of early primordial germ cells, including transcriptional regulators of human germline specification (e.g. SOX17, TFAP2C, and PRDM1). Bovine and human primordial germ cells also share expression of KIT, EPCAM, ITGA6, and PDPN genes coding for membrane-bound proteins, and an asynchronous pattern of differentiation. Additionally, the expression of members of Notch, Nodal/Activin, and BMP signaling cascades in the bovine fetal ovary, suggests that these pathways are involved in the interaction between germ cells and their niche. Results of this study provide insights into the mechanisms involved in the development of bovine primordial germ cells and put in evidence similarities between the bovine and human germline.

Eugenol influences the expression of messenger RNAs for superoxide dismutase and glutathione peroxidase 1 in bovine secondary follicles cultured in vitro.

This study aimed to investigate the effects of eugenol on growth, viability, antrum formation and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, bovine ovaries were collected from a local slaughterhouse and in the laboratory the follicles were isolated from the ovarian cortex. The follicles were then cultured in TCM-199+ alone or supplemented with different concentrations of eugenol (0.5, 5.0 and 50.0 μM). Follicular diameters and antrum formation were evaluated on days 0, 6, 12 and 18. Viability analysis was performed using calcein and ethidium homodimer. Real-time PCR was used to quantify mRNA levels for SOD, CAT, GPX1 and PRDX6 in cultured follicles. Follicular diameters and mRNA levels in follicles cultured in vitro were compared using analysis of variance and Kruskal-Wallis tests, while follicular survival and antrum formation were compared using the chi-squared test (P < 0.05). The results showed that secondary follicles cultured with eugenol maintained similar morphology and viability to follicles cultured in the control group. A progressive increase in follicular diameter was observed between days 0 and 12 for all treatments, except for follicles cultured with 50 µM eugenol. Eugenol (5.0 and 50.0 μM) increased mRNA levels for GPX1 in cultured follicles, but 0.5 μM eugenol reduced mRNA levels for SOD. The addition of eugenol did not influence mRNA expression for CAT and PRDX6. In conclusion, eugenol supplementation reduces mRNA levels for SOD and increases mRNA levels of GPX1 in bovine secondary follicles cultured in vitro.

Evidence and manipulation of O-GlcNAcylation in granulosa cells of bovine antral follicles†.

Glucose is a preferred energy substrate for metabolism by bovine granulosa cells (GCs). O-linked N-acetylglucosaminylation (O-GlcNAcylation), is a product of glucose metabolism that occurs as the hexosamine biosynthesis pathway (HBP) shunts O-GlcNAc sugars to serine and threonine residues of proteins. O-GlcNAcylation through the HBP is considered a nutrient sensing mechanism that regulates many cellular processes. Yet little is known of its importance in GCs. Here, O-GlcNAcylation in GCs and its effects on GC proliferation were determined. Bovine ovaries from a slaughterhouse, staged to the mid-to-late estrous period were used. Follicular fluid and GCs were aspirated from small (3–5 mm) and large (>10 mm) antral follicles. Freshly isolated GCs of small follicles exhibited greater expression of O-GlcNAcylation and O-GlcNAc transferase (OGT) than large follicles. Less glucose and more lactate was detectable in the follicular fluid of small versus large follicles. Culture of GCs revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase inhibitor, DON (50 μM), impaired O-GlcNAcylation and GC proliferation, regardless of follicle size. Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50 μM), also prevented proliferation, but only in GCs of small follicles. Augmentation of O-GlcNAcylation via the O-GlcNAcase inhibitor, Thiamet-G (2.5 μM), had no effect on GC proliferation, regardless of follicle size. The results indicate GCs of bovine antral follicles undergo O-GlcNAcylation, and O-GlcNAcylation is associated with alterations of glucose and lactate in follicular fluid. Disruption of O-GlcNAcylation impairs GC proliferation. Thus, the HBP via O-GlcNAcylation constitutes a plausible nutrient-sensing pathway influencing bovine GC function and follicular growth.

Indel mutations within the bovine HSD17B3 gene are significantly associated with ovary morphological traits and mature follicle number

Given the intensive selection for increased milk production, it is imperative that the problem of declining fertility in dairy cows be reversed. In female mammals their reproductive traits and functioning is controlled by a finely tuned process balancing estrogens and androgens, in which androgens (e.g., testosterone) as a precursor substance can participate in estrogen synthesis by activating 17β-hydroxysteroid dehydrogenase (17betaHSD). Being a key catalyst for testosterone synthesis, we hypothesized HSD17B3 gene is involved in the ovary’s development and thereby capable of influencing cows’ fecundity. Herein, to investigate the relationship between polymorphisms of the HSD17B3 gene and cow fertility, we characterized three insertion/deletion (indels) polymorphisms of this gene in 1110 healthy bovine ovaries. Their respective minimum allelic frequency (MAF) ranged from 0.180 to 0.482. For the ovary morphological traits, correlations revealed that both P1-D15-bp and P4-D19-bp demonstrated significant associations with ovarian height (P = 0.007 and 0.004, respectively), while P5-I5-bp was found to be significantly associated with the ovarian weight (P = 0.024). For ovarian volume, a significant correlation was uncovered between it and both polymorphisms of P4-D19-bp (P = 0.036) and P5-I5-bp (P = 0.045). Cows with either the DD genotype of P4-D19-bp or P5-I5-bp tended to have greater ovarian volume, a result consistent with their relationship to ovarian weight (P5-I5-bp) or height (P4-D19-bp). For the mature follicle traits, polymorphisms of P4-D19-bp were found significantly associated with the number of mature follicles (P = 0.045). Furthermore, expression levels of HSD17B3 differed significantly between the maximal and minimum groups of ovarian weight or volume, and the transcription factors GATA-1 and USF were predicted to bind P1-D15-bp and P4-D19-bp, respectively. This suggested the detected intron mutations could affect HSD17B3’s transcription by regulating the binding of transcription factors, thereby affecting ovarian weight and other reproductive traits. As a potential effective molecular marker loci significantly related to traits of ovary and follicle, these three indels could be used in practical molecular marker-assisted selection (MAS) breeding programs, to optimize female fertility and enhance economic efficiency in the dairy cow industry.

A loop‐shaped minimally invasive brush for improved cytology sampling of pancreatic cysts during EUS‐FNA

AbstractPancreatic cancer is the fourth leading cause of cancer‐related deaths in the West and has a 5‐year survival rate of only 2%–9%. Pancreatic cystic lesions are precursors of pancreatic cancers and a prime target for early diagnosis. Endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) enables collection of cystic fluid aiming for the diagnosis of malign and premalign cysts. However, this fluid is acellular or paucicellular in 20% of cases, hampering a proper diagnosis. Here, we introduce a minimally invasive Nitinol brush that can be operated through the 413 µm lumen of a 22G FNA needle. During operation, the brush is rotated against the inner wall of the cyst, removing cells and dispersing them in the cystic fluid, where they can be aspirated through the needle. We demonstrate the brush function using three models. An in vitro cyst model was used to visualize the brushing procedure and the mechanical interaction between the brush and the wall of a spherical cavity. Ex vivo porcine intestine and bovine ovary cyst models were used to demonstrate how brushing increases the number of harvested cells with more than one order of magnitude. These results indicate the potential of cystic brushing for the minimally invasive early diagnosis of pancreatic adenocarcinoma.

84 Characterization of agouti-signalling protein expression within the bovine ovary and early embryo

Factors present in the oocyte and surrounding follicular cells aid in the attainment of oocyte competence. Agouti-signalling protein (ASIP) is a known regulator of melanocyte function through binding to melanocortin receptors including MC1R and MC4R. Additionally, ASIP has been classified as an adipokine due to a link with insulin resistance and obesity in humans. In mice, expression is limited to hair follicles where ASIP regulates hair pigmentation. In cattle, however, ASIP mRNA has been detected in a variety of tissues, including adipose, skin, heart, testis, and the ovary. Despite ovarian expression, the role of ASIP in reproduction remains undetermined. Bovine ASIP is a secreted protein consisting of 133 amino acids. The aim of this experiment was to provide a detailed description of the ASIP expression profile within the bovine ovary and during early embryonic development. Reverse transcription PCR (RT-PCR) was conducted to analyse ASIP, MC1R, and MC4R mRNA expression. Samples examined included fetal ovaries from gestational day 90 to 250, adult ovary, fetal testis, adult testis, and 12 somatic tissues including adrenal, cerebral cortex, gut, heart, intestine, kidney, liver, lung, muscle, pituitary, stomach, and thymus. Amplification of ribosomal protein L19 (RPL19) served as a positive control for all samples. Expression of ASIP was detected in the fetal testis, 9 somatic tissues, and the fetal and adult ovary. In the fetal ovary, ASIP was detected as early as 90 days of gestation and continued throughout gestation. Expression of the ASIP receptors, MC1R and MC4R, were detected exclusively in the fetal ovary. To further characterise ASIP expression, quantitative real-time PCR (RT-qPCR) was utilised to examine samples including germinal vesicle and MII oocytes (pool of 10 oocytes), invitro-produced embryos ranging from the 2-cell to blastocyst stages (pool of 10 embryos), and cumulus and granulosa cells collected from a pool from 5 cumulus–oocyte complexes (COCs) and follicles, respectively. Theca cells from a single follicle were analysed. Samples with cycle threshold values below 35 were considered to express the gene of interest. Of the follicular cells examined, ASIP expression was present in theca, granulosa, and cumulus cells. ASIP expression was detected in both GV and MII oocytes. Early embryonic expression of ASIP was detected in the 2-cell embryo and continued to the blastocyst stage of development. In conclusion, ASIP is present in the bovine adult and fetal ovary, follicular cells including cumulus, granulosa, and theca cells, GV and MII oocytes, and invitro-produced embryos from the 2-cell to blastocyst stages. Future research will focus on identifying the function of ovarian and early embryonic ASIP in cattle.

Effect of insulin-like growth factor-I on in vitro maturation, fertilization and early embryonic development of cattle oocytes

The present experiment was conducted to investigate the effect of insulin-like growth factor-I on in vitro maturation, fertilization and early embryonic development of cattle oocytes. A total of 318 nos. bovine ovaries were collected from slaughter houses for one year and 1089 nos. culturable oocytes were collected. 357 out of 1089 nos. culturable oocytes, were subjected to IGF-I supplemented serum and serum free basic maturation media. There was no significant difference in respect of maturation, fertilization and embryonic development between IGF-I supplemented serum and serum free basic maturation as well as culture media. The mean percentage of in-vitro maturation of bovine oocytes (IVM) based on cumulus cell expansion was found to be significantly higher (P

Qualitative evaluation of slaughterhouse bovine ovaries by histological study

This study was conducted at the Anatomy and Physiology Laboratory, Faculty of Sustainable Agriculture, Universiti Malaysia Sabah Sandakan Campus from August 2016 until October 2016 to do a reach work on the analysis of the slaughterhouse bovine ovaries in term of the quality by histochemistry studies. The objectives that been set for this study were to identify the quality of follicles in bovine ovaries and to find out the relationship between the quality of follicles and ovarian state. The ovaries were collected from slaughterhouse and morphological evaluations such as weight and number of visible follicles on the surface of the ovaries were counted. The ovaries been classified into two groups as ovary with corpus luteum (CL+) and without corpus luteum (CL-). The recorded percentages of ovaries that been retrieved from the slaughterhouse is 75.0% CL- and 25.0% CL+. The visible follicles on the surface of the ovaries that been recorded at average level for CL+ (23.0 ± 2.4) is lower than the CL­(26.0 ± 2.2). From the collected ovaries represented several ovaries from both groups been used for histochemistry study. The ovaries then been prepared for histological study by fixation, dehydration, clearing, embedding, and blocking with paraffin wax. The waxed sample block been trimmed with microtome at 6µm and stained with Hematoxylene and Eosin (HE). The sample observed under microscope and the number of follicles and the granulosa cells integrity been recorded. From the finding, the CL- ovaries were contained good quality follicles (8.8 ± 0.3) at grade

152 The Effect of Nerve Growth Factor Beta on Embryonic Cleavage Rates

Abstract Nerve growth factor, (NGF) beta is a seminal plasma protein that has been associated with sire conception rates in cattle. Previous research showed that the administration of NGFβ, via culture media, to cows resulted in improved conceptus development. Though this finding was thought to be an indirect effect of improved corpus luteum (CL) function, questions raised if NGFβ could act directly on the embryo to promote development. This work seeks to determine the effect of NGFβ supplementation during in-vitro fertilization (IVF) on cleavage and blastocyst rates. How does the administration of NGFβ in culture media affect cleavage and blastocyst rates during in-vitro fertilization? Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COC). Selected COC were placed in the maturation medium. Expanded COC were inseminated with frozen-thawed spermatozoa, and IVF media was supplemented with either 0 ng/mL or 100 ng/mL NGF. Presumptive zygotes were transferred to development medium in a tri-gas chamber with 5% CO2, 5% O2, and 90% N2 in a humidified atmosphere at 39°C, mimicking the bovine uterine climate, until 8 days. Treatment with NGFβ increased the percentage of cleaved embryos at 48 hours and the percentage of hatched embryos at 8 days per oocyte. Treatment of NGFβ did not alter the percentage of blastocysts per cleaved embryo or the percentage of hatched blastocysts. These results show that the NGFβ can act directly on the embryo during fertilization to alter embryonic development, specifically embryonic cleavage rates. Future in vivo studies should assess the downstream effects of NGF treatment on conception rates in cattle.

153 The Effect of Nerve Growth Factor Beta on Embryonic Cleavage Rates

Abstract Nerve growth factor (NGF) beta is a seminal plasma protein that has been associated with sire conception rates in cattle. Previous research showed that the administration of NGFβ, via culture media, to cows resulted in improved conceptus development. Though this finding was thought to be an indirect effect of improved corpus luteum (CL) function, questions raised if NGFβ could act directly on the embryo to promote development. This work seeks to determine the effect of NGFβ supplementation during in-vitro fertilization (IVF) on cleavage and blastocyst rates. How does the administration of NGFβ in culture media affect cleavage and blastocyst rates during in-vitro fertilization? Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COC). Selected COC were placed in the maturation medium. Expanded COC were inseminated with frozen-thawed spermatozoa, and IVF media was supplemented with either 0 ng/mL or 100 ng/mL NGF. Presumptive zygotes were transferred to development medium in a tri-gas chamber with 5% CO2, 5% O2, and 90% N2 in a humidified atmosphere at 39°C, mimicking the bovine uterine climate, until 8 days. Treatment with NGFβ increased the percentage of cleaved embryos at 48 hours and the percentage of hatched embryos at 8 days per oocyte. Treatment of NGFβ did not alter the percentage of blastocysts per cleaved embryo or the percentage of hatched blastocysts. These results show that the NGFβ can act directly on the embryo during fertilization to alter embryonic development, specifically embryonic cleavage rates. Future in vivo studies should assess the downstream effects of NGF treatment on conception rates in cattle.

Bovine in vitro embryo production using media prepared with Milli-Q® Water or nanowater

The aim of this study was to compare the efficiency of in vitro embryo production (IVP) following the collection of bovine ovaries and 22-h in vitro maturation (IVM) of oocytes in media prepared with Milli-Q® Water (n = 509 oocytes) or nanowater (NW; n = 304 oocytes). The mean cleavage (63.8 ± 4.6 % vs. 63.6 ± 6.1 %, respectively; mean ± SEM) and blastocyst formation rate (16.3 ± 3.4 % vs. 16.7 ± 6.7 % of presumptive zygotes, respectively) did not vary (P > 0.05; Student t-test) between the two types of media diluents. NW is a safe substitute for Milli-Q® Water for IVM of bovine oocytes.

Effects of the mycotoxin metabolite de-epoxy-deoxynivalenol (DOM-1) on embryo development and sperm motility in cattle.

Contamination of animal feed with Fusarium spp results in accumulation of mycotoxins including deoxynivalenol. In animals, deoxynivalenol is metabolized to de-epoxy deoxynivalenol (DOM-1), which is generally considered to be a non-toxic metabolite; however, recent studies demonstrated that DOM-1 can reduce steroid production and induce apoptosis in the bovine ovary. The objectives of this study were to assess the effects of DOM-1 on applied aspects of reproductive function in cattle, specifically sperm function and embryo development in vitro and follicle growth and superovulatory responses in vivo. The effect of naturally contaminated feed on superovulatory responses was assessed; a dose of 6ppm deoxynivalenol increased blood DOM-1 concentrations to 20ng/ml, but this did not alter the number of viable embryos recovered on day 7. However, intrafollicular injection of DOM-1 (100ng/ml) directly into the growing dominant follicle resulted in cessation of follicular growth over the subsequent 3days. Treatment with DOM-1 reduced motility of bull spermatozoa over a 10-h period in vitro. Addition of DOM-1 to oocytes in vitro during IVM did not alter rates of cumulus expansion and nuclear maturation, but treatment during IVF reduced the rate of blastocyst formation. These data illustrate that DOM-1 is more biologically active than previously thought and negatively impacted reproductive outcomes in cattle.

Effect of Addition Vitamin C and Zinc Chloride in Vitrification Medium on Viability, in vitro Maturation and Ultrastructure Changes of Vitrified Immature Bovine Oocytes

This study aimed to evaluate the impact of adding vitamin C and zinc chloride to vitrification medium on viability in vitro maturation and ultrastructure changes of vitrified immature Baladi cow oocytes. Compact cumulus oocytes (COCs) (n=1370) were obtained from slaughtered bovine ovaries. Then the morphology of oocytes was examined using a stereomicroscope. Staining with trypan, the in vitro maturation and ultrastructural changes were studied. The results revealed significant (P<0.05) increase of total and normal survival rate of bovine oocytes vitrified with zinc chloride (90.28 and 81.11%) than in vitamin C media (82.5 and 65.65%) or control medium (74.44 and 54.72%). Recovery rate of abnormal bovine oocytes showed significantly an opposite trend (9.17 vs. 16.94 and 19.72%). Proportion of oocytes with viable cytoplasm and viable cumulus (VOVC) was increased significantly (93.75%, P<0.05) in fresh (control) than in medium supplemented with vitamin C and untreated medium (74.55 and 68.63%), respectively. There were non-significant differences among zinc chloride, control and vitamin C media in oocytes with vaible cytoplasm and unvaiable cumulus (VOUC). Supplementation of the vitrification medium with zinc chloride and vitamin C significantly (P<0.05) improved maturation rates (MII) of recovered cumulus oocyte complexes (COCs) than medium without supplementation. The percentage of ultrastructural alterations in most organelles bovine oocytes significantly (P<0.05) increased in oocytes vitrified without supplementation followed by vitamin C, then zinc chloride medium. Conclusion, supplementation of vitamin C or zinc chloride to the vitrification medium improved survival rate, morphologically and ultrastructural, as well as maturation rate of bovine oocytes

Molecular and cellular effects of insulin-like growth factor-1 and LongR3-IGF-1 on in vitro maturation of bovine oocytes: comparative study

Addition effects of insulin-like growth factor-1 (IGF-1) and its synthetic analogue insulin-like growth factor-1 recombinant-3 (LongR3-IGF-1) after in vitro maturation (IVM) of cattle cumulus-oocyte complexes (COCs) were compared and evaluated on meiotic progression, apoptosis and profile genes of oocyte competence (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 and IGFBP5), and their respective cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). The 739 COCs (n = 10 pools) of bovine ovaries were collected, selected and matured with IGF-1 (100 ng/mL), LongR3-IGF-1 (100 ng/mL), and in two control groups with 0.1% polyvinyl alcohol (PVA) or 10% fetal bovine serum (FBS), for 22–24 h. The statistical analysis was performed by a linear mixed effects model, ANOVA and Tukey tests. There was no statistical difference between experimental groups taken into account the meiotic progression and apoptosis (P > 0.05). Nevertheless, there were statistical differences (P ≤ 0.05) among FBS, IGF-1 and LongR3-IGF-1 groups for IGFBP4 gene expression, and among PVA, IGF-1 and LongR3-IGF-1 for COX2 gene expression in cumulus cells. Moreover, statistical difference was found for BCL2 gene expression between IGF-1, FBS and PVA groups and for IGFBP4 gene expression between LongR3-IGF-1, PVA and FBS in oocytes. There was no statistical difference between experimental groups for other genes evaluated. These results showed a good performance of IVM of bovine oocytes in the presence of LongR3-IGF-1 and the possibility of replacement of IGF-1 and FBS.

Alterations in the insulin signaling pathway in bovine ovaries with experimentally induced follicular persistence

Reproductive diseases in dairy cows as cystic ovarian disease (COD) represent a major problem that impacts on dairy production. COD is characterized by anovulation, persistence of the dominant follicle, and interruption of normal estrous cycles. Anovulation is attributable to a failure in the LH surge, due to endocrine imbalances and alterations in local factors, such as the insulin signaling pathway. Thus, the aim of this study was to examine the expression of critical nodes of the insulin pathway, including insulin receptor (IR), IR substrate (IRS), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (also known as Akt or pan-Akt), in ovarian follicular structures of cows during the development of follicular persistence induced by long-term progesterone administration. Immunoexpression of IR, p-IR, IRS1, p-IRS1, PI3K, pan-Akt and p-pan-Akt was evaluated in situ by immunohistochemistry and the concentration of insulin in serum and follicular fluid was determined by radioimmunoassay. p-IR, p-IRS1, PI3K and p-pan-Akt expression was decreased in follicles at different times of persistence in relation to the control dominant follicles, in both granulosa and theca cells, whereas IR and IRS1 immunoexpression was decreased in persistent follicles at 5 and 15 days of persistence in granulosa cells. Serum and follicular fluid insulin concentration was higher in cows with persistent follicles than in control cows. These results show that decreased expression and/or activation of the receptors and other intermediates of the insulin signaling pathway in persistent follicles indicates that reduced response/resistance to insulin rather than the concentration of insulin per se may be one of the important molecular mechanisms in the development of persistent follicles in dairy cows.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Effect of low doses of FSH and season on the in vitro maturation, fertilization and embryo development of bovine oocytes

This study was aimed at determining the effect of follicle-stimulating hormone (FSH) and season on the in vitro maturation, fertilization and embryo development of bovine oocytes. Bovine ovaries obtained from a local slaughter house were transported to the laboratory within 2–3 h in a thermos flask containing antibiotic-supplemented physiological saline (0.9%) and at a fixed temperature of 30°C. Bovine oocytes collected in spring and autumn were incubated in culture media containing FSH at concentrations of 0.2 and 0.8 μg/ml. After maturation, oocytes were fertilized. Fertilized oocytes were incubated in CR1aa culture medium for 7 days at 38.5°C for in vitro development. The assessment made after the completion of the maturation process revealed that, for both FSH doses, the maturation rates obtained with the oocytes collected in spring were higher than those obtained with the oocytes collected in autumn. The incubation of the oocytes collected in autumn in culture media supplemented with 0.2 μg/ml of FSH resulted in a low level of oocyte maturation. After maturation, oocytes were subjected to fertilization. Fertilized oocytes were incubated in CR1aa culture medium for 7 days at 38.5°C for in vitro development. In both seasons, 0.8 μg/ml FSH application was higher than the maturation values obtained with 0.2 μg/ml FSH in terms of fertilization and embryo development rates. The study was repeated 9 times for each season. Although there was no significant difference between fertilizations and embryo development in the seasons, better results were obtained in spring season.

OOCYTE SECRETED FACTORS REGULATE FSHR AND AMH mRNA LEVELS IN CUMULUS CELLS

Considering that (1) FSH activity decreases cumulus-oocyte communication via retraction of transzonal processes (TZP), (2) AMH inhibits FSH signalling by decreasing FSHR expression and adenylyl cyclase activation, and (3) the oocyte is an active player in the cumulus-oocyte complex (COC) regulating cumulus cell metabolism and gene expression via oocyte-secreted factors (OSF), we tested the hypothesis that the oocyte decreases mRNA abundance of FSHR while increasing that of AMH in cumulus cells through OSF, as part of an effort to preserve its communication with cumulus cells thus safeguarding its developmental competence. The relative mRNA abundance of FSHR and AMH in cumulus cell was compared among 3 treatment-groups: (1) Intact COC subjected to in vitro maturation (IVM); (2) oocytectomized COC (OOX) subjected to IVM and (3) OOX subjected to IVM with the addition of denuded oocytes (OOX+DO; 1 DO/μL). Four culture replicates including all three treatment-groups were performed. COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by aspiration of the ooplasm with a micromanipulator while preserving the structure of the COC. COC and OOX underwent IVM in pools of 20 for 22 hours. IVM was performed in 100 μL of TCM199 with Earl’s salts supplemented with 1μg/mL FSH, 0.4% BSA, 22 μg/mL sodium pyruvate, 75 μg/mL of amikacin at 38,5°C and 5.5% CO2 in humid atmosphere. Abundance of mRNA encoding FSHR and AMH in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and RPL15 (ribosomal protein 15) as the reference gene. Effects of treatments were tested by ANOVA and groups were compared with the Tukey test. Statistically significant differences were those with p<0.05, while p values >0.05 and <0.1 were considered as tendency of difference. Oocyctectomy decreased AMH mRNA levels (p=0.005) while tended to increase FSHR mRNA abundance (p=0.077) in cumulus cells. The addition of denuded oocytes (DO) reversed the effects of oocytectomy on both genes. Relative AMH mRNA levels (AMH/RPL15 mRNA abundance, mean ± EPM) were 0.875 ± 0.1, 0.3675 ± 0.02 and 0.7825 ± 0.1 for COC, OOX and OOX+DO, respectively. For FSHR, the respective values (FSHR/RPL15 mRNA abundance) were 1.495 ± 0.54, 2.575 ± 0.52 and 0.875 ± 0.29. Oocyte secreted factors seem to reduce FSHR expression while enhancing AMH expression in cumulus cells. We speculate that this is part of an effort of the oocyte to restrain FSH activity in the COC in order to preserve its communication with cumulus cells thus favouring developmental competence.