Interleukin-6 stimulates in vitro development of late-stage ovine embryos.

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72-192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0-192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8-16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ±â€¯1.52%) compared to the control (21.1 ±â€¯1.11%), and 5 ng/ml (25.9 ±â€¯2.98%) or 25 ng/ml (16.5 ±â€¯0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ±â€¯1.57%) and blastocyst formation (14.5 ±â€¯1.28%) compared to the control group (65.8 ±â€¯1.35% and 21.5 ±â€¯0.97%, respectively). In 8-16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

IGF-1 treatment during in vitro maturation improves developmental potential of ovine oocytes through the regulation of PI3K/Akt and apoptosis signaling.

This study aimed to assess the effect of the insulin-like grow factor 1 (IGF-1) treatment during in vitro maturation on the gene expression and developmental ability of ovine oocytes. Ovine cumulus-oocyte complexes (COC) were matured in vitro without (control) or with the supplementation of IGF-1 (100 ng/ml) and then subjected to in vitro fertilization and culture. The rate of oocyte maturation and embryo development was recorded and expression of the selected genes (involved in the PI3K/Akt and apoptosis signaling) was assessed in the matured oocytes. The IGF-1 treatment significantly (p < .05) improved the oocyte maturation rate (%) as compared to the control (81.5 ± 2.40 vs. 73.6 ± 0.94). Similarly, as compared to the control, the IGF-1 treatment significantly (p < .05) improved the rate (%) of cleavage (54.7 ± 1.58 vs. 67.2 ± 3.65) and the formation of 4-8 cell embryos (30.7 ± 2.89 vs. 44.1 ± 4.01) and morula (20.7 ± 2.08 vs. 32.8 ± 2.78). The IGF-1 treatment significantly (p < .05) upregulated the expression of IGF1R, PI3KR1, AKT1 and BCL2 and downregulated the expression of GSK3ß, FOXO3 and CASP9 in the matured oocytes. In conclusion, the IGF-1 treatment significantly improved the developmental competence of ovine oocytes through the regulation of the PI3K/Akt and apoptosis signaling.

An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos.

Assessment of intracellular reactive oxygen species (ROS) is important for evaluating the developmental ability of cumulus-oocyte complexes (COC) and embryos. Although, fluorescence-based 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method is used widely for detecting intracellular ROS in COC and embryos, it is associated with several limitations. This study aimed to develop an alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos based on nitroblue tetrazolium (NBT) staining and bright-field microscopy. Nitroblue tetrazolium reacts with ROS and forms formazan precipitate that can be detected as dark purple/blue spots under bright-field microscope. Ovine COC were matured in vitro without (control) or with the supplementation of Interleukin-7 (IL-7; for stimulating intracellular ROS), Tempol (superoxide scavenger) or combination of IL-7 and Tempol. The matured COC were stained with NBT and the formation of intracellular formazan precipitates was assessed. Additionally, the matured COC were stained with DCFH-DA to compare the level of intracellular ROS. Further, ovine embryos (8-cell, morula, and degenerating) were generated in vitro and stained with NBT for assessing intracellular ROS. The level of intracellular ROS was expressed as the proportion (%) of the NBT stained area of oocytes, compact cumulus cell masses or embryos. The proportions of NBT stained area in the matured oocytes and cumulus cells was found significantly lesser in the control as compared to the IL-7 (1 and 5 ng/ml) treated groups. A similar trend in the intracellular ROS level was also observed in the matured COC, when assessed based on the DCFH-DA staining. Following the treatment with Tempol (100 mM), negligible NBT stained area in oocytes and cumulus cells was observed. The NBT staining patterns of the oocytes and cumulus cells following the combined treatment with IL-7 (5 ng/ml) and Tempol (10 and 25 mM) were comparable with that of the control. The proportion of NBT stained area did not differ significantly between the 8-cell embryos and morula, but was found significantly greater in the degenerating embryos. In conclusion, the developed NBT staining method was found effective for detecting and interpreting the level of intracellular ROS in oocytes, cumulus cells and embryos. This method can be used as an alternative to the DCFH-DA staining method.

Reduced cytochrome oxidase activity and increased protein tyrosine phosphorylation of mitochondria-rich fractions of buffalo (Bubalus bubalis) spermatozoa after a cycle of freezing and thawing.

The motility and fertility of mammalian spermatozoa are compromised when they are cryopreserved. Sperm mitochondrial proteins play a vital role in conferring motility. However, the effects of cryopreservation on mitochondria-specific proteins remain primarily unexplored in domestic animals, including buffaloes, so the present study aimed to evaluate this issue. Mitochondria were isolated from both non-cryopreserved and cryopreserved buffalo spermatozoa by sonication followed by sucrose density gradient ultracentrifugation. The purity of the mitochondrial preparation was assessed by cytochrome oxidase assay and electron microscopy. Mitochondria separated from cryopreserved buffalo spermatozoa were associated with significantly lower (P ≤ 0.05) cytochrome oxidase activity as compared with non-cryopreserved spermatozoa. The intensities of two low-molecular-mass mitochondrial proteins (30.1 kDa and 26.1 kDa) were significantly reduced as compared with the non-cryopreserved group. In addition, in cryopreserved buffalo sperm mitochondria, the intensities of three tyrosine phosphorylated proteins (126.6, 106.7 and 26 kDa) increased significantly compared with the non-cryopreserved group. Of these, tyrosine phosphorylation of the 26-kDa mitochondrial protein of cryopreserved sperm was very intense and unique because it could not be detected in the mitochondria of non-cryopreserved sperm. Thus, the study confirmed that both cytochrome oxidase activity and the proteins of buffalo sperm mitochondria undergo significant cryogenic changes in terms of quantity and quality after a cycle of freezing and thawing and this may be one of the important causes of reduced post-thaw motility and fertility of cryopreserved buffalo spermatozoa.

Interleukin-7 improves in vitro maturation of ovine cumulus-oocyte complexes in a dose dependent manner.

Interleukin-7 (IL-7) mediated signals are linked to development, proliferation, survival and differentiation of cells. Recent evidences indicate its role in oocyte maturation process as well. Nevertheless, the underlying mechanisms of IL-7 involvement in oocyte maturation are not well characterized. In addition, currently no information is available on the effect of exogenous IL-7 on oocyte maturation in ovine or any other species. In this study, the effect of IL-7 supplementation during in vitro maturation (IVM) on the maturation rate, production of reactive oxygen species (ROS) and gene expression of ovine cumulus-oocyte complexes (COC) was assessed. IL-7 (0.5, 1, 2, 5 and 10 ng/ml) was supplemented in IVM medium at the beginning (0 h) and maturation rate of COC was assessed at the completion of IVM (24 h). The maturation rate (%) was found significantly (P = 0.000) greater with the 1 ng/ml of IL-7 supplementation (69.5) than control (60.0). In contrast, the maturation rate was reduced significantly (P = 0.000) with the 2 (47.1), 5 (39.2) and 10 ng/ml (39.1) of IL-7 as compared to the control. The level of intracellular ROS in the matured COC was found considerably higher with the 5 ng/ml of IL-7 followed by 1 ng/ml of IL-7 and control. It was evident that in the presence of superoxide dismutase-inhibitor, 1 ng/ml of IL-7 did not stimulate oocyte maturation. In contrast, oocyte maturation was improved with 5 ng/ml of IL-7 supplementation in the presence of NADPH-oxidase-inhibitor. IL-7 supplementation influenced gene expression in COC in a dose and time dependant manner. The expression of genes related to ROS production and apoptosis were upregulated and the genes associated with antioxidant mechanisms were downregulated noticeably with the supplementation of 5 ng/ml of IL-7. In conclusion, IL-7 at low concentration was beneficial for oocyte maturation, which was likely mediated through the favourable level of intracellular ROS and antioxidant mechanisms. In contrast, the detrimental effects of greater IL-7 concentrations on oocyte maturation were possibly arbitrated through the ROS-mediated oxidative stress, compromised antioxidant mechanism and stimulated apoptotic signalling.

Species-specific and differential expression of BSP-5 and other BSP variants in normozoospermic and asthenozoospermic buffalo (Bubalus bubalis) and cattle (Bos taurus) seminal plasma.

Binder of sperm-5 (BSP-5) is one of the fertility-associated proteins of cattle seminal plasma. Binding of sperm to the oviductal epithelium is mediated by BSP group of proteins. However, it is not clear, whether this protein is also involved in sperm motility. In the present study, attempts were made to characterize BSP-5 protein in both normozoospermic (NS) and asthenozoospermic (AS) Murrah buffalo (n = 18; Bubalus bubalis), Holstein Friesian (n = 8, Bos taurus) and Jersey cattle (n = 8; Bos taurus) bull seminal plasma and also study its expression pattern in these species. 1-D Western blot demonstrated three major BSP-5 immunoreactive protein bands (24.2 kDa, 20.5 kDa, and 12.3 kDa) in buffalo seminal plasma. Of these, the intensities of 24.2 and 20.5 kDa protein bands reduced significantly (P ≤ 0.05) in seminal plasma of AS group compared to that of NS group. On the contrary, the expression of 12.3 kDa protein band did not vary significantly between the groups. In Holstein Friesian seminal plasma, at least six BSP-5 immunoreactive protein bands (25.1, 23.6, 19.5, 13.8, 13.1 and 12.3 kDa) could be detected. Of these, the intensities of 23.6, 13.8/13.1 and 12.3 kDa protein bands decreased (P = 0.058, 0.111, 0.053) in AS group bulls compared to NS bulls. Holstein Friesian bull seminal plasma demonstrated a BSP-5 immunoreactive duplex protein band of 13.8/13.1 kDa, which was not evident in buffalo seminal plasma. In 2-D Western blot, a train of five BSP-5 immunoreactive duplex protein spots (Mr 21.0-27.6 kDa, pI of ∼3.9-5.1) was detected. Mass spectrometry of one of the representative duplex spot confirmed that these were BSP-5 and BSP-3 proteins, respectively. Indirect immunofluorescence studies showed that BSP-5 is primarily localized to the mid-piece/mitochondrial region of buffalo spermatozoa. To conclude, the findings of the present study could establish the significance and association of BSP-5 proteins in sperm motility and how their level differ in semen from two different clinical groups of buffalo bull (NS vs. AS). Further, the study also demonstrated that the expression pattern of BSP-5 and other BSP variants in seminal plasma of bulls is species-specific.

Temporal expression of cumulus cell marker genes during in vitro maturation and oocyte developmental competence.

PURPOSE: Cumulus cells (CC) play important roles in oocyte development and cumulus expressed genes can be used as markers for oocyte quality. This study aimed to investigate temporal changes in the expression of cumulus marker genes during oocyte maturation as possible biomarkers of embryo developmental competence in ovine. METHODS: Gene expression was assessed in the CC of the BCB+ (developmentally competent) and BCB- (developmentally poor) oocytes at 0, 12, and 24 h of in vitro maturation (IVM). Further, the association between the temporal cumulus gene expression and in vitro oocyte and embryo development was assessed. RESULTS: The maturation and blastocyst formation rates were found significantly greater for the BCB+ than the BCB- oocytes. At the 0 h of IVM, a significant upregulation in the expression of PTGS2, STAR, SDC2, LHR, FGF2, BCL2, IL7RA, HSPA1A, and IFNT was observed in the CC of the poor (BCB-) as compared to the competent (BCB+) oocytes. In contrast, it was observed that as maturation progressed, the cumulus expression of most of the favorable genes was reduced and was found significantly downregulated at the completion of IVM in the poor as compared to the competent oocytes. CONCLUSIONS: The study revealed noticeable differences in the cumulus gene expression profile at different stages of IVM between ovine oocytes of differential developmental ability. The results indicated that the loss of cumulus gene expression along the maturation period in the poor oocytes was related to their intrinsic poor quality in the ovarian follicle.

Sperm antioxidant defences decrease during epididymal transit from caput to cauda in parallel with increases in epididymal fluid in the goat (Capra hircus).

The status of antioxidant defences of both spermatozoa and their associated fluids during epididymal transit from the caput to cauda have not been studied so far in any species. Herein we report for the first time that sperm antioxidant defences, namely Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase activity, decrease significantly (P<0.05) from the caput to cauda during epididymal transit in parallel with increases in Cu,Zn-SOD, total SOD and total glutathione peroxidase (GPx) activity in the luminal fluid of the respective segments. However, levels of GPX1 and GPX3 in epididymal fluid did not change significantly from the caput to cauda. Catalase was detected for the first time in goat spermatozoa. A significantly higher total antioxidant capacity of caudal fluid than of the caput suggests a requirement for a rich antioxidant environment for the storage of spermatozoa. The retention of cytoplasmic droplets in most of the caudal spermatozoa confirmed that these droplets do not contribute to the increased antioxidant defences of cauda epididymidal fluid. Thus, the antioxidant defences of the spermatozoa and their associated epididymal fluid are modulated from the caput to cauda in a region-specific manner. This may be one of the compensatory mechanisms of epididymal fluid to scavenge any excess reactive oxygen species produced in the microenvironment of spermatozoa.

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species.

Temporal leakage of Cu,Zn superoxide dismutase and loss of two low-molecular-weight forms of glutathione peroxidase-1 from buffalo (Bubalus bubalis) sperm after freezing and thawing.

The postthaw motility and fertility of frozen-thawed buffalo spermatozoa are substantially low as compared with those of cattle sperm. The sperm motility and fertility have been positively correlated with the antioxidant enzyme activities of human and canine sperm. However, the extent of antioxidant enzyme loss during cryopreservation, although reported for human and cattle sperm, is still not clear for buffalo sperm. Thus, in the present study, an attempt was made to determine the activities of various antioxidant enzymes in buffalo spermatozoa cryopreserved for various durations (0, 30, and 60 days) and the mechanism of antioxidant enzyme loss, if any, during the process. Total superoxide dismutase (SOD) activity of cryopreserved sperm decreased and that of extended seminal plasma increased progressively with the increase in duration of cryopreservation indicating the possible time-dependent leakage of these enzymes from cryopreserved sperm into the extended seminal plasmas. The catalase and glutathione peroxidase (GPx) enzyme activities could not be detected in buffalo sperm but could be detected in fresh and extended seminal plasmas. Total GPx activities of extended seminal plasma decreased progressively with the increase in duration of cryopreservation. To confirm the presence of these enzymes at protein levels, specific antioxidant enzymes such as Cu,Zn SOD of 16 kDa and three molecular weight forms (57.7, 40.9, and 26.05 kDa) of GPx-1 were detected in buffalo sperm by Western blot. Furthermore, the intensities of 16-kDa Cu,Zn SOD in 60-day cryopreserved sperm and those of two low-molecular-weight forms of GPx-1 (40.9 and 26.05 kDa) in 30-day cryopreserved sperm decreased significantly (P < 0.05) as compared with those of noncryopreserved (0-day cryopreserved) sperm indicating selective and temporal leakage of only low-molecular-weight antioxidant proteins in the initial phase. However, all the mentioned GPx-1 forms disappeared in 60-day-old cryopreserved sperm. Immunocytochemistry experiment also revealed that Cu,Zn SOD proteins are distributed over the acrosomal region of noncryopreserved buffalo spermatozoa, and the fluorescence signal decreased substantially in 60-day cryopreserved sperm. Thus, the present study reported that there is temporal leakage of Cu,Zn SOD and loss of two low-molecular-weight forms of GPx-1 from the cryopreserved buffalo spermatozoa after freezing and thawing.

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen.

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.

Dynamic in vivo changes in the activities of gelatinases, matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases (TIMPs) in buffalo (Bubalus bubalis) uterine luminal fluid during estrous cycle and early pregnancy.

In ruminants, the phenomenon of endometrial tissue remodeling during the estrous cycle and early pregnancy is not fully understood. In this report, the occurrence of tissue remodeling, if any, in buffalo endometrium was studied by detecting gelatinases, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs); the key regulators of tissue remodeling, in uterine luminal fluids (ULF) of cycling and early pregnant (approx. 43-65 days) buffaloes. Each stage of the estrous cycle and pregnant ULF demonstrated a unique profile of gelatinase activities compared to serum/follicular fluid, with a major gelatinase band of 60 kDa with highest activity in early-luteal stage. In addition to a 32 kDa uterus-specific gelatinase band detected in both non-pregnant and pregnant ULFs, the pregnant ULF displayed three new gelatinase bands of 86, 78, and 57 kDa. Western blot technique confirmed the presence of MMP-2 (54 kDa), MMP-9 (76/73 kDa), TIMP-1 (32 kDa), TIMP-2(20 kDa), and two molecular weight forms (31 and 22 kDa) of TIMP-3 in buffalo ULF with varying band intensities. Highest MMP-2 and MMP-9 activities were observed in follicular and early-luteal stage ULFs, respectively. Highest TIMP-1 activity was observed in early-luteal ULF. Interestingly, TIMP-2 activity was only detected in mid-luteal, late-luteal, and follicular stage ULFs with significantly increasing intensities. Highest activities of 31 and 22 kDa TIMP-3 were associated with late-luteal and early-luteal stage ULFs, respectively. The varied activities of MMPs and TIMPs in buffalo ULF during the estrous cycle and early pregnancy might be a reflection of dynamic structural remodeling of the endometrium and/or developing conceptus.