DHEA restores mitochondrial dynamics of cumulus cells by regulating PGAM5 expression in poor ovarian responders.

OBJECTIVE: Poor ovarian responder (POR) poses a significant challenge for in vitro fertilization (IVF). Previous studies have suggested that dehydroepiandrosterone (DHEA) may improve IVF outcomes in POR. The current study attempts to investigate the clinical benefits of DHEA in POR and the possible mechanism of DHEA on cumulus cells (CCs). MATERIALS AND METHODS: A total of 60 women who underwent IVF treatment participated, including 22 normal ovarian responders (NORs) and 38 PORs. PORs were assigned to receive DHEA supplementation (n = 18) or not (n = 20) before IVF cycles. For all patients, CCs were obtained after oocyte retrieval. In the CCs, mRNA expression of mitochondrial dynamics relataed genes were measured. RESULTS: Supplementation of DHEA in POR reduced mitochondrial fission in CCs and decreased the expression of PGAM5 in CCs. CONCLUSION: The benefit of DHEA supplementation on IVF outcomes in POR is significant, and this effect may be mediated in part through improved mitochondrial dynamics in CC.

Evaluation of Controlled Ovarian Stimulation Protocols in Patients with Normal and Low Ovarian Reserve: Analyses of miRNAs and Selected Target Genes Involved in the Proliferation of Human Cumulus Cells and Oocyte Quality.

The oocyte and the surrounding cumulus cells (CCs) are deeply linked by a complex bidirectional cross-talk. In this light, the molecular analysis of the CCs is nowadays considered to be precious in providing information on oocyte quality. It is now clear that miRNAs play a key role in several ovarian functions, such as folliculogenesis, steroidogenesis, and ovulation. Thus, in this study, specific miRNAs, together with their target genes, were selected and investigated in CCs to assess the response of patients with normal (NR) and low (LR) ovarian reserve to two different controlled ovarian stimulation (COS) protocols, based on rFSH and hMG. Moreover, a Fourier transform infrared microspectroscopy (FTIRM) analysis was performed to evaluate DNA conformational changes in CCs and to relate them with the two COS protocols. The results evidenced a modulation of the expression of miRNAs and related target genes involved in CCs' proliferation, in vasculogenesis, angiogenesis, genomic integrity, and oocyte quality, with different effects according to the ovarian reserve of patients. Moreover, the COS protocols determined differences in DNA conformation and the methylation state. In particular, the results clearly showed that treatment with rFSH is the most appropriate in NR patients with normal ovarian reserve, while treatment with hMG appears to be the most suitable in LR patients with low ovarian reserve.

Exposure to Pulsed Electromagnetic Fields Improves the Developmental Competence and Quality of Somatic Cell Nuclear Transfer Buffalo (Bubalus bubalis) Embryos Produced Using Fibroblast Cells and Alters Their Epigenetic Status and Gene Expression.

We examined the effects of treatment with pulsed electromagnetic fields (PEMFs) on cumulus cells and buffalo somatic cell nuclear transfer (SCNT) embryos. PEMF treatment (30 µT for 3 hours) of cumulus cells increased (p < 0.05) the relative cell viability and cell proliferation and the expression level of OCT4, NANOG, SOX2, P53, CCNB1, and GPX, but decreased (p < 0.05) that of DNMT1, DNMT3a, GSK3b, and BAX, whereas the expression level of DNMT3b, GLUT1, BCL2, CASPASE3, SOD1, and CATALASE was not affected. PEMF treatment of SCNT embryos at the beginning of in vitro culture increased (p < 0.05) the blastocyst rate (51.4% ± 1.36% vs. 42.8% ± 1.29%) and decreased (p < 0.01) the apoptotic index to the level in in vitro fertilization blastocysts, but did not significantly alter the total cell number and the inner cell mass:trophectoderm cell number ratio of blastocysts compared to the controls. PEMF treatment increased the expression level of NANOG, SOX2, CDX2, GLUT1, P53, and BCL2 and decreased that of BAX, CASPASE3, GSK3b, and HSP70, but not OCT4, DNMT1, DNMT3a, DNMT3b, HDAC1, and CCNB1 in blastocysts. It increased (p < 0.001) the global level of H3K27me3 but not H3K18ac. These results suggest that PEMF treatment of SCNT embryos improves their developmental competence, reduces the level of apoptosis, and alters the expression level of several important genes related to pluripotency, apoptosis, metabolism, and stress.

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

Peroxiredoxin 1 Controls Ovulation and Ovulated Cumulus-Oocyte Complex Activity through TLR4-Derived ERK1/2 Signaling in Mice.

Peroxiredoxins (PRDXs) are expressed in the ovary and during ovulation. PRDX1 activity related to the immuno-like response during ovulation is unknown. We investigated the roles of Prdx1 on TLR4 and ERK1/2 signaling from the ovulated cumulus-oocyte complex (COC) using Prdx1-knockout (K/O) and wild-type (WT) mice. Ovulated COCs were collected 12 and 16 h after pregnant mare serum gonadotropin/hCG injection. PRDX1 protein expression and COC secretion factors (Il-6, Tnfaip6, and Ptgs2) increased 16 h after ovulated COCs of the WT mice were obtained. We treated the ovulated COCs in mice with LPS (0.5 µg/mL) or hyaluronidase (Hya) (10 units/mL) to induce TLR4 activity. Intracellular reactive oxygen species (ROS), cumulus cell apoptosis, PRDX1, TLR4/P38/ERK1/2 protein expression, and COC secretion factors' mRNA levels increased in LPS- and Hya-treated COCs. The ERK inhibitor (U0126) and Prdx1 siRNA affected TLR4/ERK1/2 expression. The number and cumulus expansion of ovulated COCs by ROS were impaired in Prdx1 K/O mice but not in WT ones. Prdx1 gene deletion induced TLR4/P38/ERK1/2 expression and cumulus expansion genes. These results show the controlling roles of PRDX1 for TLR4/P38/ERK1/2 signaling activity in ovulated mice and the interlink of COCs with ovulation.

A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells.

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

Upregulation of Low-Density Lipoprotein Receptor of the Steroidogenesis Pathway in the Cumulus Cells Is Associated with the Maturation of Oocytes and Achievement of Pregnancy.

The maturation of the oocyte is influenced by cumulus cells (CCs) and associated with pregnancy rate, whereas the influencing factors have not been completely elucidated in the CCs. In this study, we identified new regulators of CCs for high-quality oocytes and successful pregnancies during assisted reproductive techniques. CCs were collected from cumulus-oocyte complexes (COCs) in young (≤33 years old) and old (≥40 years old) women undergoing intracytoplasmic sperm injection (ICSI) procedures. We screened for factors differentially expressed between young vs. old CCs and pregnancy vs. non-pregnancy using whole mRNA-seq-next-generation sequencing (NGS). We characterized the transcriptome of the CCs to identify factors critical for achieving pregnancy in IVF cycles. Women in the young and old pregnancy groups exhibited the up- and downregulation of multiple genes compared with the non-pregnancy groups, revealing the differential regulation of several specific genes involved in ovarian steroidogenesis in CCs. It was shown that the low-density lipoprotein (LDL) receptor to the steroidogenesis pathway was upregulated in CCs with higher maturity rates of oocytes in the pregnancy group. In conclusion, a higher pregnancy rate is related to the signaling pathway of steroidogenesis by the LDL receptor in infertile women undergoing IVF procedures.

Differential Transcript Profiles in Cumulus-Oocyte Complexes Originating from Pre-Ovulatory Follicles of Varied Physiological Maturity in Beef Cows.

Small dominant follicle diameter at induced ovulation, but not at spontaneous ovulation, decreased pregnancy rate, fertilization rate, and day seven embryo quality in beef cows. We hypothesized that the physiological status of the follicle at GnRH-induced ovulation has a direct effect on the transcriptome of the Cumulus-Oocyte complex, thereby affecting oocyte competence and subsequent embryo development. The objective of this study was to determine if the transcriptome of oocytes and associated cumulus cells (CC) differed among small (≤11.7 mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7-14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). RNA sequencing data, from pools of four oocytes or their corresponding CC, revealed 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte pools from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. An additional 128, 98, and 80 DEG were identified among small versus large, small versus spontaneous, and large versus spontaneous follicle CC pools, respectively. The biological pathway "oxidative phosphorylation" was significantly enriched with DEG from small versus spontaneous follicle oocyte pools (FDR < 0.01); whereas the glycolytic pathway was significantly enriched with DEG from CC pools obtained from large versus small follicles (FDR < 0.01). These findings collectively suggest that altered carbohydrate metabolism within the Cumulus-Oocyte complex likely contributes to the decreased competency of oocytes from small pre-ovulatory follicles exposed to an exogenous GnRH-induced gonadotropin surge.

In vivo dynamic 3D imaging of oocytes and embryos in the mouse oviduct.

Developmental biologists have always relied on imaging to shed light on dynamic cellular events. However, processes such as mammalian fertilization and embryogenesis are generally inaccessible for direct imaging. In consequence, how the oviduct (fallopian tube) facilitates the transport of gametes and preimplantation embryos continues to be unanswered. Here we present a combination of intravital window and optical coherence tomography for dynamic, volumetric, in vivo imaging of oocytes and embryos as they are transported through the mouse oviduct. We observed location-dependent circling, oscillating, and long-distance bi-directional movements of oocytes and embryos that suggest regulatory mechanisms driving transport and question established views in the field. This in vivo imaging approach can be combined with a variety of genetic and pharmacological manipulations for live functional analysis, bringing the potential to investigate reproductive physiology in its native state.

In vitro maturation using an agarose matrix with incorporated extracellular matrix proteins improves porcine oocyte developmental competence by enhancing cytoplasmic maturation.

Here, we present a novel in vitro maturation (IVM) system comprising an agarose matrix supplemented with extracellular matrix (ECM) proteins for enhanced maturation of immature oocytes within cumulus-oocyte complexes (COCs) derived from porcine medium antral follicles (MAFs). Immunocytochemical analyses of integrin subunit α2 , α5 , α6 , ß1 , and ß4 expression suggested that integrin α2 ß1 , α5 ß1 , α6 ß1 , and α6 ß4 play pivotal roles in IVM of porcine immature oocytes. Combinatorial supplementation of fibronectin interacting with integrin α5 ß1 , collagen interacting with integrin α2 ß1 , and laminin interacting with integrin α6 ß1 and α6 ß4 to the agarose matrix had no significant effect on nuclear maturation. However, the number of parthenogenetic embryos that developed into blastocysts increased when oocytes were matured using agarose IVM matrices supplemented with fibronectin, collagen, or laminin. Furthermore, significant increases in cytoplasmic maturation-related parameters (BMP15 level, cumulus cell expansion score, intra-oocyte ATP level, and index of cortical granule distribution) were observed in COCs matured in vitro using ECM protein-incorporated agarose matrices. Our data suggest that mature porcine oocytes with enhanced developmental competence and high-quality cytoplasm can be generated via IVM using agarose matrices supplemented with fibronectin, collagen, or laminin.

Effects of Early Cumulus Cell Removal on Treatment Outcomes in Patients Undergoing In Vitro Fertilization: A Retrospective Cohort Study.

Background: Early cumulus cell removal combined with early rescue intracytoplasmic sperm injection (ICSI) has been widely practiced in many in vitro fertilization (IVF) centers in China in order to avoid total fertilization failure. However, uncertainty remains whether the pregnancy and neonatal outcomes are associated with early cumulus cell removal. Objectives: To investigate if early cumulus cell removal alone after 4 hours co-incubation of gametes (4 h group), has detrimental effect on the pregnancy and neonatal outcomes in patients undergoing IVF, through a comparison with conventional cumulus cell removal after 20 hours of insemination (20 h group). Methods: This retrospective cohort study included 1784 patients who underwent their first fresh cleavage stage embryo transfer at the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital from June 2016 to December 2018 (4 h group, n=570; 20 h group, n=1214). A logistic regression analysis was performed to examine the independent association between early cumulus cell removal and pregnancy outcomes after adjustment for potential confounders. The neonatal outcomes between the two groups were compared. Results: When compared with the 20 h group, the 4 h group had similar pregnancy outcomes, including rates for biochemical pregnancy, clinical pregnancy, ongoing pregnancy, miscarriage, ectopic pregnancy, multiple pregnancy, live birth. There were 1073 infants delivered after embryo transfer (4 h group, n=337; 20 h group, n=736). Outcomes in both groups were similar for both singleton and twin gestations, including preterm birth rate and very preterm birth rate, mean birth weight, mean gestational age, sex ratio at birth and rate of congenital birth defects. In addition, findings pertaining to singleton gestations were also similar in the two groups for Z-scores (gestational age- and sex-adjusted birth weight), rates of small for gestational age, very small for gestational age, large for gestational age and very large for gestational age infants. Conclusions: In this study early cumulus cell removal alone was not associated with adverse pregnancy and neonatal outcomes. From this perspective, early cumulus cell removal to assess for a potential early rescue ICSI is therefore considered to be a safe option in patients undergoing IVF.

Hydroxyurea affects in vitro porcine oocyte maturation through increased apoptosis and oxidative stress.

Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus-oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.

Lipid Metabolism in Bovine Oocytes and Early Embryos under In Vivo, In Vitro, and Stress Conditions.

Lipids are a potential reservoir of energy for initial embryonic development before activation of the embryonic genome and are involved in plasma membrane biosynthesis. Excessive lipid droplet formation is detrimental to cryotolerance and is related to alterations in mitochondrial function, which likely affects lipid metabolism. Increased lipid accumulation in in vitro produced embryos is a consequence of the stress during in vitro embryonic development process. There are several open questions concerning embryo lipid metabolism and developmental potential. Oocyte maturation and embryo development in vivo and in vitro may vary if the donors are subjected to any type of stress before follicle puncture because crucial changes in oocyte/embryonic metabolism occur in response to stress. However, little is known about lipid metabolism under additional stress (such as heat stress). Therefore, in this review, we aimed to update the information regarding the energy metabolism of oocytes and early bovine embryos exhibiting developmental competence, focusing on lipid metabolic pathways observed under in vivo, in vitro, and stress conditions.

Embryonic Environmental Niche Reprograms Somatic Cells to Express Pluripotency Markers and Participate in Adult Chimaeras.

The phenomenon of the reprogramming of terminally differentiated cells can be achieved by various means, like somatic cell nuclear transfer, cell fusion with a pluripotent cell, or the introduction of pluripotency genes. Here, we present the evidence that somatic cells can attain the expression of pluripotency markers after their introduction into early embryos. Mouse embryonic fibroblasts introduced between blastomeres of cleaving embryos, within two days of in vitro culture, express transcription factors specific to blastocyst lineages, including pluripotency factors. Analysis of donor tissue marker DNA has revealed that the progeny of introduced cells are found in somatic tissues of foetuses and adult chimaeras, providing evidence for cell reprogramming. Analysis of ploidy has shown that in the chimaeras, the progeny of introduced cells are either diploid or tetraploid, the latter indicating cell fusion. The presence of donor DNA in diploid cells from chimaeric embryos proved that the non-fused progeny of introduced fibroblasts persisted in chimaeras, which is evidence of reprogramming by embryonic niche. When adult somatic (cumulus) cells were introduced into early cleavage embryos, the extent of integration was limited and only cell fusion-mediated reprogramming was observed. These results show that both cell fusion and cell interactions with the embryonic niche reprogrammed somatic cells towards pluripotency.

Development of in-vitro maturation protocol for rat oocytes; under simple culture vs co-culture with cumulus cell monolayer and its developmental potential via Parthenogenetic/artificial activation.

BACKGROUND: Murine is the most abundantly used as laboratory animal models. There has been a tremendous amount of research including; their evolution, growth, physiology, disease modeling as well as genomic mapping. Rats and mice are the most widely used among them. Although both rats and mice fall under the same category still both are different a lot too. As regarding in vitro maturation and development mouse studies are well established as compared to rats which still lies in the early phase of development. So, we tried to figure out rat oocytes in vitro maturation and their developmental potential by performing 3 experiments i.e. superovulation, in vitro Maturation as simple culture (COC's only), and COC's & cumulus cells co-culture, which later further developed using parthenogenetic activation after IVM. Female Sprague Dawley rat 3-4 week used for these studies, we hyper-stimulated their ovaries using PMSG and hCG 150 IU/kg each. After that, we collected ovaries via dissection and retrieved oocytes. We matured them in TCM 199 supplemented with FSH, Estrogen, EGF, and Pyruvate. After maturation, we activated them using two types of activators i.e. Ethanol 7%, Ionomycin. After that, we saw and compared their developmental potential in vitro. RESULTS: Oocytes matured in COC's and Cumulus cell monolayer co-culture (59% ± 4*) showed significantly more even growth and extrusion of the first polar body as compared to the COC's only culture (53.8 ± 7%*). While oocytes activated using Ionomycin showed more promising development until 8 cells/blastocyst level compared to ethanol 7%. CONCLUSION: we concluded that COC's and cumulus monolayer co-culture is better than COC's only culture. Cumulus monolayer provides extra aid in the absorption of nutrients and supplements thus providing a better environment for oocytes growth. Also, we concluded that matured oocytes showed more developmental capacity after activation via ionomycin compared to ethanol.

Human bone morphogenetic protein 8A promotes expansion and prevents apoptosis of cumulus cells in vitro.

Cumulus expansion is essential for ovulation and oocyte maturation in mammals. Previous studies suggest that this process requires certain cumulus expansion enabling factors, induced by LH surge, that activate SMAD signaling locally. However, their identities remain uncertain. Using a superovulated rat model, we showed that Bmp8 transcripts were abundant in cumulus cell-oocyte complexes (COCs) and their levels can be further induced during ovulation. By analyzing human COC-related transcriptomic datasets, BMP8 transcripts in cumulus cells were also found to be significantly elevated along with the maturation status and developmental competence of enclosed oocytes. In cultured rat COCs, treatment with recombinant BMP8A protein activated both SMAD1/5/8 and SMAD2/3 pathways; the resulting SMAD2/3 signaling induced COC expansion as well as the expression of COC expansion-related genes, whereas the resulting SMAD2/3 and SMAD1/5/8 activations were both required for protecting expanded cumulus cells from apoptosis. Taken together, our data demonstrated that addition of BMP8 protein in the in vitro rat COC cultures not only promotes cumulus expansion but also sustains survival of expanded cumulus cells via different SMAD downstreams. With these capabilities, BMP8 may have clinical applications to ameliorate the fertilizability and subsequent developmental competence of the enclosed oocytes when doing in vitro COC maturation.

Optimization and Comparison of Three-Dimensional Culture Conditions in Different Media of Coculture and Encapsulation System for In Vitro Follicular Development in Bubalus bubalis.

The establishment of an in vitro culture system for complete oocyte maturation from the early stages of ovarian follicles is still a challenge. The aim of the present study was to assess the effect of different matrix with different culture media on the developmental growth of ovarian follicles in vitro. An ovarian histoarchitectural study was carried out to identify the primordial (0.027-0.039 mm), primary (0.041-0.079 mm), small preantral (0.085-0.131 mm), large preantral (0.132-0.294 mm), small antral (0.387-0.589 mm), and large antral (1.188-1.366 mm) follicles. Thus, large preantral follicles (0.2-0.3 mm) were mechanically isolated and cultured subsequently in different microconditions such as Dulbecco's modified Eagle's medium, Tissue Culture Medium-199 (TCM-199) and Opti-minimum essential medium, with same supplements where control (without matrix) was compared with matrix (coculture and encapsulation), which includes (1) buffalo fetal fibroblast cells, (2) cumulus cells, (3) ovarian mesenchymal cells, (4) collagen, (5) gelatin, and (6) Matrigel, cultured for 7 days in CO2 incubator at 38.5°C (5% CO2 in air). Cultured follicles were evaluated for growth rate (107.88% ± 10.24%), maturation rate (51.06% ± 6.53%), survivability rate (56.52% ± 3.42%), and antioxidant (catalase; CAT [1.58 ± 0.04 U/mg], superoxide dismutase; SOD [4.63 ± 0.05 U/mg], lactate dehydrogenase; LDH [1.48 ± 0.01 U/mg]) enzymatic activities, which showed significantly (p < 0.05) positive results in growth model with media TCM-199 than other studied groups. Furthermore, the development of large preantral follicles augmented significantly (p < 0.05) for growth rate (248.54% ± 9.51%), maturation rate (75.81% ± 7.07%), survivability rate (81.82% ± 3.02%), antioxidant (CAT [2.05 ± 0.03 U/mg], SOD [3.13 ± 0.12 U/mg], LDH [2.55 ± 0.51 U/mg]), and estradiol (175.83 ± 5.92 pg/mL) activities when they were encapsulated in Matrigel with nutritional requirements fulfilled by media TCM-199. These results provide better insight for the optimization of culture conditions for in vitro follicular development in the water buffalo, which will eventually assist in resolving the limitation of obtaining fewer competent oocytes for the embryo production in the species.

In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes.

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5-0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.

The advanced paraffin-section preparation technique based on multiple cumulus-oocyte complexes rather than ovaries in ovine.

Immunohistochemical staining is the important method for the identification of protein expression in mammal ovaries, in particular in the follicles with the potential to develop into cumulus-oocyte complexes (COCs), which are able to support oocyte maturation regardless of in vivo or in vitro. Here, we reported an advanced immunohistochemical method based on an artificial structure gathering multiple COCs by paraffin embedding for rapid and highly sensitive detection of co-expressed proteins in ovine COCs rather than ovaries. Compared with the conventional immunohistochemistry on ovine ovaries, the advanced COC paraffin sectioning technique showed the better immunostaining effect and featured the higher generation rate for COCs, the distincter cumulus layers, and the more simplified procedures. These results indicate that the COC paraffin sectioning technique is highly effectively applied for identification of protein expression in ovine COC.

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.