Evaluation of ImageJ software in ultrasonic image analysis: Follicular and luteal morphological characteristics of cattle.

This study was conducted to compare the effectiveness of two methodologies in evaluating B- and Doppler-mode ultrasonic images: analysis using ultrasonic software and utilizing a computer with ImageJ software. To determine if ImageJ software utilization is an efficacious alternative to ultrasonic software device- analysis, there were comparisons of values when using the two methods for morphological and vascular characteristics of follicular dynamics and luteal function in 18 crossbred cattle. From day 8 of an ovarian dynamics synchronization treatment regimen period until the time of ovulation (Day 10), B-mode and power-flow ultrasonic cineloops were obtained every 12 h to assess follicular diameter, wall area, and wall blood perfusion area. On Day 14 after ovulation, US cineloops of ovaries were obtained in B mode and power flow to evaluate various morphological and vascular characteristics of the corpus luteum (CL), including luteal diameter, luteal area, and CL blood perfusion area. Cineloops were evaluated and analyzed using ultrasonic software, and in a computer with ImageJ software. To evaluate consistency in results between the two methods, there was evaluation utilizing paired t-test, Pearson correlation coefficient, Bland-Altman plot, and Linear Regression Test procedures to calculate proportion of bias between values for measurements of variables evaluated. Results indicated none of the values for variables before and after ovulation differed (P > 0.05). It, therefore, was concluded that utilization of ImageJ software is an efficacious biomedical technique to analyze ultrasonic images of morphological and vascular characteristics before and after ovulation in cattle.

Luteinizing Hormone Regulation of Inter-Organelle Communication and Fate of the Corpus Luteum.

The corpus luteum is an endocrine gland that synthesizes the steroid hormone progesterone. luteinizing hormone (LH) is a key luteotropic hormone that stimulates ovulation, luteal development, progesterone biosynthesis, and maintenance of the corpus luteum. Luteotropic and luteolytic factors precisely regulate luteal structure and function; yet, despite recent scientific progress within the past few years, the exact mechanisms remain largely unknown. In the present review, we summarize the recent progress towards understanding cellular changes induced by LH in steroidogenic luteal cells. Herein, we will focus on the effects of LH on inter-organelle communication and steroid biosynthesis, and how LH regulates key protein kinases (i.e., AMPK and MTOR) responsible for controlling steroidogenesis and autophagy in luteal cells.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Melatonin modulates swine luteal and adipose stromal cell functions.

Based on our previous study in follicles, the first aim of this work was to evaluate the effect of melatonin in the swine corpus luteum (CL). Luteal cells were exposed to 10 and 20pg mL-1 melatonin. We evaluated the effect on proliferation (bromo-deoxy-uridine uptake), steroidogenesis (progesterone) and redox status by means of Griess test (nitric oxide production), WST-1 test (superoxide anion generation) and FRAP test (non-enzymatic antioxidant power). The results showed a significant increase in antioxidant power, as well as a reduction in the other parameters analysed. These data and the expression of MT2 observed in luteal cells allow us to hypothesise a physiological role of melatonin in the regulation of CL functionality. The reproductive function is dependent on energy reserves stored in adipose tissue. Therefore, we sought to verify the effect of melatonin on adipose stromal cells (ASCs). MT2 receptor expression was detected in ASCs and the presence of gene markers (PPARγ and leptin) before and after adipogenic differentiation was verified. The differentiation was significantly inhibited by melatonin, as well as cell viability. In conclusion, present results suggest that melatonin exerts a potential inhibitory action on luteal function and adipogenesis, possibly mediated by MT2.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice.

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.

Novel Insights on the Corpus Luteum Function: Role of Vaspin on Porcine Luteal Cell Angiogenesis, Proliferation and Apoptosis by Activation of GRP78 Receptor and MAP3/1 Kinase Pathways.

Formation and limited lifespan of corpus luteum (CL) are important for proper ovarian periodicity and fertility. Failed vascularization, imbalance between proliferation and apoptosis leads to luteal phase deficiency and infertility. The aim of this study was to examine the effect of vaspin on angiogenesis, apoptosis and proliferation as well as the involvement of 78-kDa glucose-regulated protein receptor (GRP78) and mitogen-activated kinase (MAP3/1) in these processes. Porcine luteal cells were incubated with vaspin (0.1-10 ng/mL) for 24 h to 72 h and then mRNA and protein expression of angiogenesis: vascular endothelial growth factor (VEGFA), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), VEGFA receptors (VEGFR1, VEGFR2), apoptosis: caspase 3, bcl-2-like protein 4 (BAX), B-cell lymphoma (BCL2), and proliferation: proliferating cells nuclear antigen (PCNA), cyclin A factors as well as secretion of VEGFA, FGF2, ANGT1 were measured by real-time polymerase chain reaction (PCR), immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Moreover, apoptosis was assessed by caspase activity using the Caspase-Glo 3/7 assay, while proliferation was by alamarBlue. We found that vaspin enhanced luteal cell angiogenesis, proliferation, and significantly decreased apoptosis. Additionally, using GRP78 siRNA and the pharmacological inhibitor of MAP3/1 (PD98059), we observed that the effect of vaspin was reversed to the control level in all investigated processes. Taken together, our results suggest that vaspin is a new regulator of female fertility by direct regulation of CL formation and maintenance of luteal cell function.

Responses to intra-luteal administration of cloprostenol in dairy cows.

The aim of the study was to determine the luteolytic dose of cloprostenol administered directly into the corpus luteum (CL; intra-luteal treatment, ILT) in dairy cattle. Cows of two control groups were treated with 500 µg of cloprostenol (Estrumate®) intramuscularly (IM-500) or via ILT with 0.2 mL of physiological solution (ILT-0). Cows of four experimental groups were treated by ILT with cloprostenol in doses 5, 25, 50 and 100 µg (ILT-5, -25, -50 and -100 groups). Progesterone concentrations (P4) and size of CL were evaluated to assess luteolysis at 0, 0.5, 1, 2, 4, 8, 24 and 48 h or at 0, 24 and 48 h after ILT, respectively. Cows in the ILT-0 and -5 groups were unaffected by ILT. The P4 concentrations were less in cows of the IM-500, as well as ILT-25, -50 and -100 groups at 48 h subsequent to ILT. The size of the CL was less in cows of IM-500, as well as ILT-25, -50 and -100 groups at 48 h after ILT. There were P4 concentrations of about 1 ng/mL 48 h after ILT in cows of the IM-500, as well as ILT-50 and -100 groups. In conclusion, the cloprostenol dose of 50 µg administered intra-luteally is a luteolytic dose in cows.

Pentraxin-3 mediates prosurvival actions of interferon tau in bovine luteinized granulosa cells.

Pentraxin 3 (PTX3), a multimeric glycoprotein, is implicated in various biological functions. PTX3 was shown to be elevated in the corpus luteum (CL) of early pregnant ewes; however, its role in sheep or other ruminants' CL during this reproductive stage or how it is regulated remain unknown. Here we explored the role of PTX3 and its relationship with interferon-tau (IFNT; the pregnancy recognition signaling molecule during early pregnancy in domestic ruminants) in bovine luteinized granulosa cells (LGCs). IFNT robustly elevated PTX3 expression in bovine LGCs, and significantly stimulated its expression in luteal endothelial cells, along with CL slices; yet, LGCs were the most responsive and sensitive among these luteal models. ALK2/ALK3/ALK6 kinase inhibitor, dorsomorphin, dose-dependently inhibited basal and IFNT-elevated PTX3 expression in LGCs. In contrast, ALK4/5/7 inhibitor, SB431542, did not alter basal and TGFB1-induced PTX3. We found that recombinant human PTX3 itself moderately but significantly increases LGC numbers. Because PTX3 is highly expressed in bovine LGCs, we next examined the impact of lowering endogenous PTX3 levels with siRNA. PTX3 silencing decreased the viable cell numbers and reversed IFNT actions on cell viability, percentage of proliferating cells, and on two key survival/death genes: BIRC5 encoding surviving protein, and FASL - a death-inducing signal. Interestingly, thrombospondin-1, a known luteal proapoptotic factor, was inversely related to PTX3 in LGCs. Together, these findings suggest a novel role for PTX3 during early pregnancy, as mediator of IFNT prosurvival actions supporting CL maintenance during this reproductive stage.

Gene Expression Profiling of Corpus luteum Reveals Important Insights about Early Pregnancy in Domestic Sheep.

The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (e.g., TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (SIGLEC3, SIGLEC14, SIGLEC8), ribosomal proteins (RPL17, RPL34, RPS3A, MRPS33) and chemokines (CCL5, CCL24, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized non-coding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated TXNL1 gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including TXNL1, SIGLEC14, SIGLEC8, MRP4, and CA5A).

Clustered microRNAs: The molecular mechanism supporting the maintenance of luteal function during early pregnancy.

MicroRNAs (miRNAs) are recognized as the important regulators of ovarian function. However, little is known about the hormonal regulation of miRNA expression and the role of the specific miRNA-mRNA interactions in corpus luteum. Therefore, the present study was undertaken to determine: (a) the expression of miRNAs in the corpus luteum in early pregnancy vs regression; (b) the effect of conceptus and uterine signals in the expression of selected miRNAs; and (c) the role of specific miRNA-mRNA interactions in the molecular changes and secretory function of the corpus luteum in the pig. The results showed that the majority of miRNAs differentially expressed in the corpus luteum in early pregnancy vs regression belong to independent clusters (eg, miR-99b, miR-532), which are highly conserved among different animal species. The main conceptus signal in the pig (17ß-estradiol) elevated the luteal expression of the miR-99b cluster and lowered the expression of NR4A1 and AKR1C1, the genes involved in corpus luteum regression. Furthermore, the delivery of miR-99b cluster mimics to luteal tissue concomitantly decreased NR4A1 and AKR1C1 expression and enhanced progesterone secretion. The present study demonstrated that conceptus signals can support the maintenance of luteal function during pregnancy by clustered miRNA-stimulated pathways, governing the expression of genes involved in luteal regression.

Effects of recombinant bovine somatotropin on pregnancy per artificial insemination, corpus luteum cellular composition and endometrial gland morphometry in beef cattle.

The aim of this research was to evaluate the effect of recombinant bovine somatotropin (bST) on pregnancy per artificial insemination (P/AI), cellular composition of the corpus luteum (CL) and endometrial gland morphometry. In Experiment 1, Nelore cows (n = 587) received a fixed-time artificial insemination (FTAI) protocol and, at insemination, received 0, 250 or 500 mg of bST subcutaneously (SC). In Experiment 2, Nelore cows (n = 243) received 0 or 500 mg of bST, SC, on D7 (D0 = day of FTAI). Blood samples were collected on D7 and D16 to measure progesterone (P4) concentrations. In Experiments 1 and 2, pregnancy diagnosis was performed 30 days after FTAI. In Experiment 3, Nelore heifers (n = 20) received a FTAI protocol, but were not inseminated, and on D0 (ovulation day), they received 0 (bST 0; n = 9) or 500 mg of bST (bST 500; n = 11), SC. The heifers were slaughtered on D15 (D0 = ovulation day), at which time the CL was evaluated for diameter, weight, a percentage of large (LLC) and small (SLC) luteal cells, and the concentration of progesterone in plasma measured. The number, perimeter and area of superficial and deep endometrial glands were evaluated. There was no difference in P/AI when bST was applied on D0 and D7. In Experiment 1, P/AI did not differ among treatments, with 59.28% (115/194), 58.38% (115/197) and 65.82% (129/196) for the bST 0, 250 and 500 treatments, respectively. In Experiment 2, P/AI did not differ between treatments, with 57.3% (71/124) and 60.5% (62/119) for the bST 0 and 500 treatments, respectively. Plasma progesterone concentrations on D16 was greater in the bST 500 (11.63 ±â€¯0.84 ng/mL) than bST 0 (9.83 ±â€¯0.88 ng/mL). In Experiment 3, there was no difference in ovarian diameter and weight, CL diameter, percentage of SLC, P4 concentrations and endometrial gland morphology. Heifers in the bST 500 treatment had heavier CL (3.11 ±â€¯0.32 vs. 2.25 ±â€¯0.20 g); however, the bST 0 treatment heifers had a greater percentage of LLC than did the bST 500 treatment (13.72 ±â€¯1.16% vs. 8.60 ±â€¯1.52). It was concluded that the doses of bST used in this study do not increase P/AI; however, they do cause changes in P4 concentration and the cellular composition of the CL.

Effects of prostaglandin F2α (PGF2α) on cell-death pathways in the bovine corpus luteum (CL).

BACKGROUND: Prostaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF2α may depend on its local vs. systemic actions. In our study, we determined changes in expression of genes related to: (i) apoptosis: caspase (CASP) 3, CASP8, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and (ii) necroptosis: receptor-interacting protein kinase (RIPK) 1, RIPK3, cylindromatosis (CYLD), and mixed lineage kinase domain-like (MLKL) in the early and mid-stage corpus luteum (CL) that accompany local (intra-CL) vs. systemic (i.m.) analogue of PGF2α (aPGF2α) actions. Cows at day 4 (n = 24) or day 10 (n = 24) of the estrous cycle were treated by injections as follows: (1) systemic saline, (2) systemic aPGF2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF2α (2.5 mg; Dinoprost). After 4 h, CLs were collected by ovariectomy. Expression levels of mRNA and protein were investigated by RT-q PCR, Western blotting and immunohistochemistry, respectively. RESULTS: We found that local and systemic administration of aPGF2α in the early-stage CL resulted in decreased expression of CASP3 (P < 0.01), but CASP8 mRNA expression was up-regulated (P < 0.05). However, the expression of CASP3 was up-regulated after local aPGF2α treatment in the middle-stage CL, whereas systemic aPGF2α administration increased both CASP3 and CASP8 expression (P < 0.01). Moreover, we observed that both local and systemic aPGF2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL (P < 0.05) while CYLD expression was markedly higher after i.m. aPGF2α injections (P < 0.001). Moreover, we investigated the localization of necroptotic factors (RIPK1, RIPK3, CYLD and MLKL) in bovine CL tissue after local and systemic aPGF2α injections in the bovine CL. CONCLUSION: Our results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF2α administration depending on its local or systemic actions. Locally-acting PGF2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF2α-induced luteolysis in cattle.

Early pregnancy diagnosis at 21 days post artificial insemination using corpus luteum vascular perfusion compared to corpus luteum diameter and/or echogenicity in Nelore heifers.

The objective was to compare the use of corpus luteum (CL) vascular perfusion to CL diameter and/or echogenicity to diagnose pregnancy at 21 d after timed-AI. Ovaries of Nelore heifers were assessed using ultrasonography in B-mode and color Doppler simultaneously 21 d after timed-AI (n = 113). Objective evaluations were performed using an image processing software to extract the number of colored pixels (ColorPix), diameter (mm) and echogenicity/mm² (EchoPix) of the CL. Subjective evaluations of the CL were performed by five evaluators using scores of estimated vascular perfusion area of color Doppler scan videos and estimated CL size and qualitative echogenicity of B-mode scan videos. The reference pregnancy diagnosis was performed 33 d after timed-AI using an ultrasonic device. Corpus luteum ColorPix, diameter and EchoPix were highly correlated (P < 0.001) with pregnancy. Pregnancy diagnosis accuracy, sensitivity, and negative predictive value were not different for CL ColorPix and diameter and was less with use of EchoPix compared to the other parameters. Size and vascular perfusion scores were correlated to the greatest extent (0.88-0.94) with the respective objective values within evaluator. The results from the ROC curve analysis indicated a smaller area under the curve for qualitative echogenicity compared to CL size and vascular perfusion. Corpus luteum vascular perfusion was the only subjective evaluation that when assessed there were no false negative pregnancy diagnoses. In conclusion, the use of the objective CL diameter resulted in the same efficacy as CL vascular perfusion evaluations for early pregnancy diagnosis in Nelore heifers.

An objective volumetric method for assessment of ovarian follicular and luteal vascular flow using colour Doppler ultrasonography.

Our goal was to develop an objective computer-assisted volumetric method of assessing vascular flow from colour Doppler ultrasound data of ovarian structures recorded by free-hand movement. We hypothesized that a vascularity index (ratio of the region of blood flood to the region of ovarian structure) obtained from the three-dimensional volumetric analysis would be more precise (less variable) than conventional two-dimensional analysis of single images in estimating the functional status of the preovulatory follicles and corpus luteum. Doppler ultrasound cineloops of water buffaloes (Bubalus bubalis; n = 22) ovaries were recorded daily from 12 h before GnRH treatment to four days after ovulation. Cineloops were processed using Fiji and Imaris software packages for segmenting the area (two-dimensional analysis) and the volume (three-dimensional analysis) occupied by the blood-flow and associated tissue to calculate the vascularity index. For volumetric measurement, all images in a cineloop were used (i.e., no a-priori selection of images) while for two-dimensional analysis, three images from the region with apparent maximum vascularity were selected. The volumetric method was verified with theoretical ellipsoidal volume of the follicle (r = 0.96 P < 0.01) or corpus luteum (r = 0.58 P = 0.02). The variability in the follicular vascularity index among animals was lower using the volumetric method than two-dimensional analysis (0.018 ±â€¯0.002 vs 0.030 ±â€¯0.005, P < 0.01), while the variability for CL vascularity was similar between methods (P = 0.23). An increase in the follicular vascularity index was detected at 12 h after GnRH treatment using both methods (two-dimensional: 0.030 ±â€¯0.008, P < 0.01; three-dimensional: 0.016 ±â€¯0.006, P < 0.02). Buffaloes that ovulated tended to have a greater increase in 3D vascularity index than non-responding buffaloes (P = 0.06); the two-dimensional method was not able to detect these changes. Using the three-dimensional method, a moderate positive correlation (r = 0.59; P = 0.02) was evident between the follicular vascularity index at 14-16 h after GnRH treatment and follicular diameter. In conclusion, an objective volumetric method for assessing relative ovarian blood flow changes was developed using Doppler ultrasound cineloops recorded by free-hand movement. The 3-dimensional method eliminates the need for a-priori selection of images and is more precise as a result of decreased technical variability.

Lipopolysaccharide disrupts gap junctional intercellular communication in an immortalized ovine luteal endothelial cell line.

Gram-negative bacteria, in particular Escherichia coli with its cell wall lipopolysaccharide (LPS), often cause metritis and mastitis in domestic animals. Ovarian LPS accumulation may initiate local inflammatory reactions mediated through cell surface Toll-like receptors (TLRs). This may disrupt ovarian functionality leading to infertility. Possible adverse effects of LPS on luteal activity are not yet well explored. We hypothesized that LPS could lead to alterations in luteal vascular functionality. Therefore, we established an in vitro cell line model (OLENDO) by immortalizing microvascular endothelial cells isolated from ovine corpus luteum (CL) with a potent Simian Virus 40 T-antigen (SV40-Tag). OLENDO exhibit endothelial cell characteristics, like low-density lipoprotein (LDL) uptake, express BSL-I, and VEGFR2, as well as TLR2 and TLR4 receptors. LPS-treatment of OLENDO altered in vitro tube formation, had no effects on cell viability and decreased gap junctional intercellular communication (GJIC). LPS did not impair GJA1/Cx43 protein expression, but altered its cellular localization showing signs of internalization. Taken together, we demonstrated the mechanisms underlying LPS induced impairment of luteal GJIC and immune processes in a novel and well-characterized OLENDO cell line.

Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96-182 cluster miRNAs in endothelial cells.

Our previous studies showed that the miRNA clusters, miR-183-96-182 and miR-212-132, may be critical in promoting luteal cell survival and progesterone production in both bovine and humans. To further understand their involvement in luteal development, this study aimed to establish the expression of these miRNAs in different bovine luteal cell types, namely, endothelial and steroidogenic, isolated using fluorescence-activated cell sorting (FACS). We isolated each of the two cell populations based on the presence of the endothelia surface marker, CD144, and uptake of the lipophilic dye, Nile Red, respectively. Using quantitative Polymerase Chain Reaction (qPCR) in the sorted cell fractions we confirmed that CD144 and the endothelia-specific miRNA, miR-126, were predominantly expressed in endothelial cells (CD144+), whereas HSD3B1 was expressed predominantly in steroidogenic cells (Nile RedHI). Finally, we found that whereas the miR-212-132 cluster was expressed at similar levels in luteal endothelial and steroidogenic cells, miR-183-96-182 was expressed at > 4-fold higher levels in endothelial than in steroidogenic cells (P < 0.05), suggesting that these two miRNA clusters, and particularly miR-183-96-182, may be important in functionally regulating not only steroidogenic cells but also endothelial cells in the corpus luteum (CL).

A potential function of RLIP76 in the ovarian corpus luteum.

Ral interacting protein of 76 kDa (RLIP76) is multifunctional protein localized and distributed in the plasma membrane, cytosol, and nucleus of the cell. In tumorigenesis, RLIP76 emerges as a common feature for the solid tumor growth. RLIP76 is ubiquitously expressed in various tissues including the ovary. Interestingly, the similar physiological events in obtaining an adequate supply of nutrient by gaining access to the host vascular system are required either for corpus luteum formation or tumor development. In addition, the identical angiogenesis modulators were found in neoplastic and normal ovaries. Our previous study involving RLIP76-/- mice implanted with melanoma or carcinoma cell conclusively demonstrated that RLIP76 is necessary for angiogenesis and neovascularization of primary solid tumors. RLIP76 plays an essential role in tumor angiogenesis through the regulation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1). In certain previous studies, those pro-angiogenic factors were found significantly to be upregulated during the corpus luteum formation. To that, the following review will discuss the likelihood of RLIP76 role in ovarian corpus luteum.

Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.