The follicular microenvironment in low (++) and high (I+B+) ovulation rate ewes

Reproduction ◽  
2020 ◽  
Vol 159 (5) ◽  
pp. 585-599
Author(s):  
Zaramasina L Clark ◽  
Derek A Heath ◽  
Anne R O’Connell ◽  
Jennifer L Juengel ◽  
Kenneth P McNatty ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Single Copy ◽  
Oocyte Quality ◽  
Ovulation Rate ◽  
Fluid Composition ◽  
Embryo Survival ◽  
Expression Levels ◽  
Follicular Maturation ◽  
Metabolic Substrates

Ewes with single copy mutations in GDF9, BMP15 or BMPR1B have smaller preovulatory follicles containing fewer granulosa cells (GC), while developmental competency of the oocyte appears to be maintained. We hypothesised that similarities and/or differences in follicular maturation events between WT (++) ewes and mutant ewes with single copy mutations in BMP15 and BMPR1B (I+B+) are key to the attainment of oocyte developmental competency and for increasing ovulation rate (OR) without compromising oocyte quality. Developmental competency of oocytes from I+B+ animals was confirmed following embryo transfer to recipient ewes. The microenvironment of both growing and presumptive preovulatory (PPOV) follicles from ++ and I+B+ ewes was investigated. When grouped according to gonadotropin-responsiveness, PPOV follicles from I+B+ ewes had smaller mean diameters with fewer GC than equivalent follicles in ++ ewes (OR = 4.4 ± 0.7 and 1.7 ± 0.2, respectively; P < 0.001). Functional differences between these genotypes included differential gonadotropin-responsiveness of GC, follicular fluid composition and expression levels of cumulus cell-derived VCAN, PGR, EREG and BMPR2 genes. A unique microenvironment was characterised in I+B+ follicles as they underwent maturation. Our evidence suggests that GC were less metabolically active, resulting in increased follicular fluid concentrations of amino acids and metabolic substrates, potentially protecting the oocyte from ROS. Normal expression levels of key genes linked to oocyte quality and embryo survival in I+B+ follicles support the successful lambing percentage of transferred I+B+ oocytes. In conclusion, these I+B+ oocytes develop normally, despite radical changes in follicular size and GC number induced by these combined heterozygous mutations.

117 Subclinical Mastitis Reduces Ovulation and Oocyte Quality in Milk-Producing Cows

2018 ◽  
Vol 30 (1) ◽  
pp. 198
Author(s):  
G. Santos ◽  
M. P. Bottino ◽  
M. B. D. Ferreira ◽  
J. C. Silveira ◽  
A. C. F. C. M. Avila ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Target Genes ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Subclinical Mastitis ◽  
Oocyte Quality ◽  
Ovulation Rate ◽  
Follicular Dynamics

The aim was to evaluate the effect of subclinical mastitis by somatic cell count (SCC) on follicular dynamics, ovulation, oocyte and cumulus cell quality, exosome size and concentration in milk-producing cows. Crossbred cows (Bos taurus × Bos indicus; that is, Holstein × Gyr) were randomly allocated to control (SCC <200,000 cells mL−1] and mastitis (SCC >400,000 cells mL−1) groups. In experiment 1 (follicular dynamics), cows (n = 57) were submitted to ultrasonographic evaluations every 24 h, after removal of an intravaginal progesterone device (Day 8) up to Day 10. From Day 10, ultrasound evaluations were performed every 12 h, until ovulation or until 96 h after progesterone device withdrawal, in order to follow final dominant follicle growth and ovulation. In experiment 2 (oocyte, cumulus cells, and follicular fluid evaluation), cows (n = 23) were submitted to follicular aspirations, preceded by synchronization of the emergence of the follicular wave. The levels of target genes in cumulus cells (BCL2, BAX, PI3K, PTEN, FOXO3) were evaluated by RT-qPCR. In the follicular fluid, the exosomes were isolated for evaluation of particle size. Data were analysed by the Glimmix procedure of SAS (SAS Institute Inc., Cary, NC, USA). Ovulation rate (P = 0.09) was higher in control cows [control 77.42% (24/31) and mastitis 57.69% (15/26)]. Viable oocyte rate (P = 0.01) was also higher in control cows [control 59.1% (130/220) and mastitis 41.9% (125/298)]. The dynamics of follicular growth did not differ between groups. The number of degenerate oocytes (P = 0.001) was higher in cows of the mastitis group. In the evaluation of cumulus cell gene expression, there was a higher abundance of BAX transcripts (P = 0.003) in cells of mastitis cows. Additionally, the mean and mode of exosome diameter in mastitis cows were smaller (P = 0.03 and P = 0.02, respectively). In conclusion, ovulation rate, oocyte quality, and follicular fluid exosome diameter were lower in cows with subclinical mastitis, demonstrating a link between mammary gland sanitary status and reproduction.

126 Interference of mastitis with ovulation and oocyte and granulosa cell quality in dairy cows

2019 ◽  
Vol 31 (1) ◽  
pp. 188
Author(s):  
G. Santos ◽  
M. P. Bottino ◽  
A. P. C. Santos ◽  
R. E. Orlandi ◽  
L. M. S. Simões ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Follicular Fluid ◽  
Rate Control ◽  
Cumulus Cell ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Subclinical Mastitis ◽  
Oocyte Quality ◽  
Ovulation Rate ◽  
Intravaginal Device

The objective of this study was to evaluate the effect of mastitis diagnosed by somatic cell count (SCC) on follicular growth, ovulation, oocytes and cumulus cells quality and the concentration and size of exosomes in follicular fluid of dairy cows. In the study, crossbred cows (Bos taurus-Holstein×Bos indicus-Gir) were classified for analysis as control (SCC &lt;200.000 cells mL−1) and mastitis (SCC &gt;400.000 cells mL−1) groups. In Experiment 1 (follicular dynamics), cows (n=57: control=31; mastitis=26) received a progesterone intravaginal device (Sincrogest®, Ourofino Saude Animal, Cravinhos, Brazil) and 2mg of oestradiol benzoate (Sincrodiol®, Ourofino Saude Animal) injected IM. Eight days later (D8), the progesterone device was removed and cows received IM 500mg of cloprostenol (Sincrocio®, Ourofino Saude Animal), 1mg of oestradiol cypionate (SincroCP®, Ourofino Saude Animal) and 300IU of eCG (SicroeCG®, Ourofino Saude Animal). Ultrasound exams (Mindray 4900, probe linear de 5MHz, Shenzhen, China) were performed every 24h from removal of the progesterone-releasing intravaginal device (D8) until 48h later. Thereafter, evaluations were performed every 12h, until ovulation or up to 96h after removal of the progesterone-releasing intravaginal device. In Experiment 2 (oocyte, cumulus complexes, and follicular fluid evaluation), cows (n=26: control=13; mastitis=13) were submitted to follicular aspiration (ovum pickup) for oocyte quality and cumulus cells transcript evaluation. Transcript abundance of apoptosis markers (BCL2, BAX, PI3K, PTEN, FOXO3) was determined by real-time RT-PCR. Moreover, 7 days after the ovum pickup session, the dominant follicle was aspirated and follicular fluid samples were obtained. Exosomes were isolated from the follicular fluid by serial centrifugations, which were also performed for evaluation of particle size and concentration. Statistical analyses were performed using the SAS (SAS Institute Inc., Cary, NC, USA), and the GLIMMIX procedure was used to determine significant differences between groups. Gene expression and exosome data were submitted to the Student’s t-test. Ovulation rate [control 77.4% (24/31) and mastitis 57.7% (15/26); P=0.09] and viable oocytes rate [control 59.1% (130/220) and mastitis 41.9% (125/298); P=0.01] were higher in control animals. Additionally, there was a greater number of degenerate oocytes (control 6.7±1.2 and mastitis 13.3±5.5; P=0.001) in subclinical mastitis cows. There was greater abundance (P=0.003) of BAX cumulus cell transcripts and exosome mean (P=0.03) was smaller in subclinical mastitis cows. However, BCL2, PI3K, PTEN, nd FOXO3 cumulus cell transcripts was similar between treatments. In conclusion, ovulation rate, oocyte quality, and exosome diameter were smaller in cows with SCC &gt;400.000 cells mL−1, demonstrating that subclinical mastitis can influence the fertility of dairy cows.

scholarly journals Oocytes in sheep homozygous for a mutation in bone morphogenetic protein receptor 1B express lower mRNA levels of bone morphogenetic protein 15 but not growth differentiation factor 9

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Janet L Crawford ◽  
Derek A Heath ◽  
Karen L Reader ◽  
Laurel D Quirke ◽  
Norma L Hudson ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Cumulus Cell ◽  
Ovulation Rate ◽  
Mrna Levels ◽  
Camp Production ◽  
Expression Levels ◽  
Bone Morphogenetic Protein Receptor ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Validation Experiment

The aim of this study was to test the hypothesis that the high ovulation rate in ewes (BB) homozygous for a mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene is linked to lower BMP15 and/or GDF9 mRNA in oocytes compared with those in wild-type (++) ewes. Cumulus cell–oocyte complexes (COC) and granulosa cells (GC) were recovered from ≥1 mm diameter follicles of BB and ++ ewes during a prostaglandin-induced follicular phase. Expression levels of GDF9 and BMP15 were measured by multiplex qPCR from individual COC. The gonadotropin-induced cAMP responses of the GC from each non-atretic follicle were measured following treatment with FSH or human chorionic gonadotropin. In a separate validation experiment, GDF9 and BMP15 expression was present only in oocytes and not in cumulus cells. There was no effect of follicular diameter on oocyte-derived GDF9 or BMP15 mRNA levels. The mean expression levels of BMP15, but not GDF9, were significantly lower in all non-atretic follicles, including the subsets containing either FSH- or LH-responsive GC in BB, compared with ++, ewes. No genotype effects were noted for FSH-induced cAMP production by GC either with respect to dose of, or number of follicles responding to, FSH. However, ovaries from BB ewes contained significantly more follicles responsive to LH, with respect to cAMP production in GC. We propose that these findings are consistent with the hypothesis that the higher ovulation rate in BB sheep is due, at least in part, to lower oocyte-derived BMP15 mRNA levels together with the earlier onset of LH-responsiveness in GC.

scholarly journals Next-Generation Sequencing Reveals Downregulation of the Wnt Signaling Pathway in Human Dysmature Cumulus Cells as a Hallmark for Evaluating Oocyte Quality

2020 ◽  
Vol 1 (3) ◽  
pp. 205-215
Author(s):  
Ryosuke Akino ◽  
Daisuke Matsui ◽  
Ryouka Kawahara-Miki ◽  
Mitsuyoshi Amita ◽  
Kuniko Tatsumi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Expression Patterns ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Next Generation ◽  
Expression Levels ◽  
Morphological Evaluation ◽  
Generation Sequencing

Background: Dysmature cumulus cells are lower fertilization rates and abnormalities in embryonic development compared to maturation cumulus cells. Morphological evaluation of cumulus–oocyte complexes (COCs) considered the possibility that differences may also be found in gene expression. Purpose: To identify hallmarks for evaluating oocyte quality by investigating gene expression patterns in human cumulus cells surrounding oocytes. Methods: Cumulus cells were obtained from the cumulus–oocyte complex of infertile women treated with assisted reproductive technology. Based on maturity level, the cumulus cells were classified into two categories, i.e., dysmature cumulus cell (DCC) and maturation cumulus cell. DCCs were subjected to gene expression analysis using next-generation sequencing and compared with COCs that are in the process of maturation as controls. Results: The expression levels of genes involved in the Wnt signal/β-catenin pathway were significantly reduced in DCCs compared with those in control cells. Moreover, the expression levels of genes involved in multiple pathways associated with apoptosis were also significantly reduced compared with those in control cells. Conclusions: DCCs showed significant decreases in apoptosis- and Wnt/β-catenin signaling-associated gene expression. DCCs could be classified by morphological evaluation, and the method described in this study may be useful as an oocyte quality estimation tool.

96 Follicular Fluid and Serum Biochemical Composition in Alpacas (Vicugna pacos) with Different Nutritional Planes

2018 ◽  
Vol 30 (1) ◽  
pp. 187
Author(s):  
W. Huanca ◽  
F. Hilari ◽  
M. Ticona ◽  
B. Lira ◽  
J. C. Villanueva ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Biochemical Composition ◽  
Oocyte Quality ◽  
Metabolizable Energy ◽  
High Rate ◽  
Hypodermic Needle ◽  
Limited Information ◽  
Embryo Survival ◽  
Natural Pasture ◽  
Vicugna Pacos

The importance of nutrition on composition of follicular fluid and oocyte quality is recognised in several species but limited information is available on alpacas and would explain the high rate of early embryo mortality. The aim of this study was to analyse the biochemical composition of serum and follicular fluid (FF) from alpacas (Vicugna pacos) with different nutritional planes. Twelve adult female alpacas between 6 and 8 years old were assigned randomly to either a high (n = 6) or low (n = 6) plane of nutrition. Nutritional planes were defined by the grazing condition of the natural pasture with (high) or without (low) supplementation with 200 g of concentrate/day (total digestible nutrients: 52%, protein: 16%, and metabolizable energy: 2.8 Mcal kg−1). The nutritional conditions were imposed 1 month before the start of the experiment.The concentrations of total glucose, cholesterol, triglycerides, and proteins were determined with a semi-automatic biochemical analyzer (SINOWA, China). When an ovulatory follicle ≥7 mm was detected by ultrasonography using a 5-MHz linear-array transducer (SSD-500, Aloka, Tokyo, Japan), a transvaginal transducer with an attached needle guide (UST-945BP-5, Aloka) was used to collect follicular fluid. Follicular puncture was performed using a disposable 19-gauge # 12 mm hypodermic needle connected to a 50-mL conical tube via a silicon tube. A caudal epidural anaesthesia was induced with 4 mL of 2% lidocaine. Blood samples were obtained by jugular venipuncture into serum vacutainers and kept at room temperature for 30 min. Follicular fluid and serum were centrifuged at 1500 × g for 20 min, decanted, and stored at –20°C until analysed. Results were analysed by Student’s t-test. The results are presented in Table 1. The results suggest differences in biochemical composition of glucose and cholesterol in follicular fluid, which could explain the oocyte quality and embryo survival in alpacas. Table 1.Biochemical composition of follicular fluid (FF) and serum from alpacas (Vicugna pacos) under 2 nutritional planes Research was supported by INNOVATE PERU Grant 405-PNICP-PIAP-2014.

Relationships between follicular fluid composition and follicular/oocyte quality in the mare

1999 ◽  
Vol 60 (2-3) ◽  
pp. 243-253 ◽  
Author(s):  
Nadine Gérard ◽  
Guy Duchamp ◽  
Michèle Magistrini
Keyword(s):  
Follicular Fluid ◽  
Oocyte Quality ◽  
Fluid Composition

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

Follicular environment as a predictive tool for embryo development and kinetics in cattle

2019 ◽  
Vol 31 (3) ◽  
pp. 451 ◽  
Author(s):  
Gláucia Pereira Alves ◽  
Fernanda Bertuccez Cordeiro ◽  
Camila Bruna de Lima ◽  
Kelly Annes ◽  
Érika Cristina dos Santos ◽  
...  
Keyword(s):  
Embryo Development ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Large Scale ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Fluid Composition ◽  
Predictive Tool ◽  
Transcription Pattern ◽  
Transcriptional Pattern

Follicular fluid composition and the transcription pattern of granulosa cells were analysed to better comprehend associations between embryo development and morphokinetics. Bovine follicles were punctured and their respective follicular fluid and granulosa cells were collected. Cumulus–oocyte complexes derived from these follicles were matured and fertilised invitro. Embryo morphology and kinetics were evaluated at 40h after insemination, when embryos were classified as fast (FCL, four or more cells), slow (SCL, 2–3 cells) or non-cleaved (NCL). Their development was followed until the blastocyst stage. Glucose, pyruvate, cholesterol and oestradiol were quantified in the follicular fluid and the transcription pattern of 96 target genes was evaluated in granulosa cells by large-scale quantitative reverse transcription polymerase chain reaction. Follicular fluid from the blastocyst group had increased levels of glucose, total cholesterol and pyruvate compared to the non-blastocyst group, whereas higher levels of oestradiol were observed in the follicular fluid of embryos and blastocysts with fast cleavage. The transcriptional pattern revealed altered metabolic pathways between groups, such as lipid metabolism, cellular stress and cell signalling. In conclusion, both follicular fluid and granulosa cells are associated with the possibility of identifying follicles that may generate embryos with high potential to properly develop to the blastocyst stage.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

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