SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing.     

Kininase II from human seminal plasma. Isolation and properties

A rapid and highly efficient procedure for purification of kininase II from human seminal plasma is described. After ultra centrifugation, the enzyme was purified by gel filtration on Sepharose 6B CL and ion exchange chromatography followed by affinity chromatography of EDTA-inhibited enzyme on bradykinin-Sepharose. The enzyme was specifically inhibited by Captopril and BPP9a but not by phosphoramidon. PAGE in the presence of sodium dodecyl sulfate under reducing conditions resulted in two major protein bands with apparent molecular masses of about 55 kDa and 65 kDa and two faint protein bands at higher molecular masses. Antibodies raised against the major protein bands showed full cross reactivity with all four protein bands. The presented data indicate that kininase II consists of subunits.     

MicroRNA-related markers associated with corpus luteum tropism in buffalo (Bubalus bubalis)

The study was undertaken to decipher the microRNA (miRNA) related markers associated with corpus luteum (CL) tropism in buffalo. The data obtained from deep sequencing of CL tissue from different physiological stages was mined in silico for the identification of miRNA-related markers (SSR & SNP). From the present study, 5 annotated and 176 unannotated miRNA were deduced while comparing with Bos taurus genome. In addition, 4 SSRs and 9 SNPs were deduced from the miRNA sequences. These SSRs were on the genes viz. Eukaryotic translation initiation factor 1-like, myocyte enhancer factor 2A, beta casein, T cell receptor gamma cluster 1. The SNP positions on genes viz. PYGO1 (Pygopus family PHD finger 1), LOC100337244 (Multidrug resistance-associated protein 4), FTH1 (Ferritin heavy chain 1), LOC788634 (BOLA class I histocompatibility antigen), PLXND1 (Plexin D1) and UBC (Ubiquitin C) show that these genes play critical role in CL tropism during estrous cycle in buffalo.     

Comparative analysis of protein profiles of wild virulent (E156) and aroA-htrA double deletion mutant vaccine strain (S30) of Salmonella enterica subsp. enterica serovar Abortusequi under in vivo and in vitro growth conditions

In the present study, cell lysate and cell supernatant of the both strains i.e., virulent wild type (E156) and mutant (S30) vaccine strains of Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), grown under varied in vivo and in vitro conditions were subjected to SDS PAGE and western blotting (using rabbit hyperimmune serum). Variation in growth conditions did not have any significant effect on expression of different proteins. SDS PAGE of E156 and S30 cell lysate (CL) revealed 26 and 28 bands, respectively with 3 prominent proteins of 71, 46 and 42 kDa in cell lysate of E 156 and 4 prominent proteins 71, 65, 46 and 40 kDa in S30 strain. The cell supernatant (CS) from both the strains, subjected to SDS PAGE, exhibited similarity in protein profile among these strains, however three bands of 65, 53 and 40 kDa were more prominent in CS preparation of S30, whereas a 56 kDa protein was prominent in CS of E156. Western blotting of E156 and S30 revealed 3 unique proteins of 65, 53 and 40 kDa present in CS preparation of S30 strains which could be used for differentiation of mutant and wild strains and also in development of test for differentiating vaccinated animals from naturally infected.     

Factors affecting number of surface ovarian follicles and oocytes yield and quality in Egyptian buffaloes

Three experiments were conducted to evaluate factors affecting number of surface ovarian follicles and oocytes yield and quality in buffalo. In Experiment 1, ovaries (n = 126) were collected in pairs from slaughtered anoestrus, early pregnant and cyclic buffaloes. Ovarian follicles (1-3, 4-9 and > or = 10 mm diameter) were counted, aspirated and oocytes were recovered and evaluated. In Experiment 2, ovaries were divided into 2 groups. Group 1, ovaries bearing a CL (n = 74) and Group 2 non-bearing CL (n = 74), ovarian follicles (2-8 mm) were counted, aspirated and oocytes evaluated. In Experiment 3, oocytes were recovered using aspiration or slicing methods. In all experiments, oocytes were classified into good, fair, poor and denuded. Results showed that the development of small and total ovarian follicles are continuous and independent in early pregnant or cyclic buffalo cows, however, it significantly decreased (P < 0.01) in the ovaries of anoestrus buffaloes. Number of medium and large size follicles was significantly increased (P < 0.01) in cyclic buffaloes on Days 10-16 and 17-22 of oestrous cycle, while large follicles was significantly decreased (P < 0.01) in the ovaries of pregnant buffaloes. A significantly higher (P < 0.01) percentage of poor and denuded oocytes were recovered from ovaries of anoestrus and pregnant buffalo. While, the highest (P < 0.01) percentage of good quality oocytes were recovered from ovaries of cyclic buffaloes on Days 1-3 and 10-16 of oestrous cycle, eliciting that the stage of oestrous cycle is affecting the quality of buffalo oocytes. In addition, the presence of a CL stimulates the development of a significantly higher (P < 0.01) number ovarian follicles which produced a significantly higher (P < 0.05) number of good quality oocytes. Slicing of buffalo ovaries produced a significantly higher number of fair, poor and denuded oocytes. In conclusion, number of ovarian follicles and yield and quality of oocytes were affected by the reproductive status, stage of the oestrous cycle, presence of a CL and the method of oocytes retrieval.     

Concentrations of catecholamines, ascorbic acid, progesterone and oxytocin in the corpora lutea of cyclic and pregnant cattle.

To determine if there are inter-relationships between progesterone, oxytocin (OT), dopamine (DA), noradrenaline (NA) and ascorbic acid, these compounds were measured in the corpus luteum (CL) from cattle at different stages of the oestrous cycle (n = 42) and from 1-5 months of pregnancy (n = 27). They were measured by radioimmunoassay (RIA), high performance liquid chromatography (HPLC) and colorimetric methods. Corpora lutea were collected from heifers and cows within 30 min of slaughter on days 1-5, 6-10, 11-16 and 17-21 of the oestrous cycle. The stage of pregnancy was determined on the basis of foetal size and development. Each CL was divided into four parts and stored in liquid nitrogen. For hormone estimation, the tissue was homogenised/powdered and suspended in phosphate buffer (for OT and progesterone), 0.1 M trichloracetic acid (TCA; for catecholamines) or in ice-cold metaphosphoric acid (for ascorbic acid). There were no significant differences in the measured parameters between cows and heifers, and so the data were combined. The concentration of DA was correlated with NA (r = 0.66; P < 0.001) during the oestrous cycle and was highest in newly formed CL (P < 0.01) as compared with early CL, regressed CL and CL of pregnant females. NA was negatively correlated (P < 0.01) with progesterone (r = -0.53) and OT (r = -0.41). In contrast, progesterone and OT were positively correlated with each other (r = 0.81; P < 0.01) during all stages of the oestrous cycle, but not during pregnancy. The lowest concentrations of ascorbic acid were observed in regressed CL. Ascorbic acid concentrations were correlated (P < 0.01) with those of progesterone (r = 0.68), OT (r = 0.42) and DA (r = -0.37). Luteal concentrations of ascorbic acid, progesterone and OT followed a pattern consistent with the development and regression of the CL. Luteal concentrations of catecholamines were not consistent with this pattern.     

Multiple Laminin Binding Proteins in Human Fetal Heart

The possibility of occurrence of laminin binding proteins in cardiac tissue under different stages of growth was examined by affinity chromatography of the soluble fraction of human fetal myocardial plasma membrane over Ln-Sepharose. A 67 kDa protein was isolated by elution with glycine/HCl buffer containing 1 M NaCl and visualized as a coomassie stainable band on SDS gel electrophoresis under reducing conditions. Dot blot assays of the radioiodinated protein revealed the binding of 67 kDa protein with high affinity to laminin in a cation independent manner. This protein appears to be present in relatively higher amounts in tissues from early stage fetus. The occurrence of cation dependent laminin binding proteins was also examined by affinity chromatography. Electrophoresis of the EDTA eluate under reducing conditions followed by silver staining showed two prominent bands with average molecular size 130 and 174 kDa which under non-reducing conditions appeared as two bands with average molecular weight of 115 and 135 kDa. Using radioiodinated protein in dot blot assays, its binding to Ln was found to be maximum in the presence of Mn⁺⁺ ions. Immunoblotting using anti-β₁ integrin antibodies showed that 115 kDa protein is a β₁ integrin suggesting the possibility of this protein belonging to the integrin group of receptors. The occurrence of multiple laminin binding proteins and the relative abundance of one of these proteins viz. the 67 kDa protein during early stages than in late stage tussue suggest a possible role for these proteins in cellular interactions with laminin during myocardial tissue development.     

Kinetic Changes of Nucleic Acids and Protein During Embryogenesis of Wheat (Triticun vulgaris L.)

Per embryonic total nucleic acid, RNA content and per cell RNA content increased during embryogenesis, reached maximun at 21 day after anthersis. The per embryo and per cell protein content also increased concomitantly. But the protein content continued to increase up to 24 days after anthersis. On the basis of dry weight, RNA content decreased in the early stage of embryogenesis, but then increased over the period of later developmental stage. The protein content on the basis of dry weight also changed in similar way. It was likely the protein and RNA content changes concomitantly during the developmental process of wheat embryo. As to per embryo DNA content, it increased in early developmental stage, but then remained in a similar level during the later stage. The relationship between the changes of RNA content and protein synthesis, embryonie develope is also discussed in present paper.     

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.     

Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa

Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS–PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 μg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29 ± 2.7, 2.61 and 0.2 mg/ml, respectively. Eighteen total protein bands were observed in the range of 12–127 kDa. Eight major HB proteins were isolated in the range of 13–71 kDa. Seven major GB proteins were isolated in the range of 13–61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9 ± 0.6) followed by GB proteins (25.4 ± 0.6) and control (21.2 ± 1.4). The difference in BCMPT values between protein treated and control group was significant (P < 0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4 ± 0.65 and 66.1 ± 0.6, respectively). The difference in HOST values between proteins treated and control group (50.4 ± 2.0) was significant (P < 0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin–sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 μg/ml gave best results) of buffalo cauda spermatozoa.     

Proliferation of Luteal Steroidogenic Cells in Cattle

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages.     

Phospholipid and nucleic acid gradients in the developing amphibian embryo

Rana pipiens embryos at the end of the blastula stage were dissociated and the cell suspension was separated into presumptive ectoderm, mesoderm, light endoderm, and heavy endoderm cells by a discontinuous density gradient centrifugation technique. The isolated germ layers were analyzed for total lipid, lipid phosphorus, plasmalogen, RNA, and DNA. Per gram dry weight, DNA showed a threefold decrease from ectoderm to heavy endoderm. On the same basis, the RNA content of the mesoderm was 34 per cent higher than that of ectoderm, and 320 and 570 per cent higher than that of light and heavy endoderm, respectively. In addition to the RNA and DNA gradients, there were at least two superimposed lipid gradients: a neutral lipid gradient decreasing from ectoderm to endoderm, and a total phospholipid gradient increasing from ectoderm to endoderm. In contrast to total phospholipid, a specific phospholipid class, ethanolamine plasmalogen, decreased from ectoderm to endoderm. The total lipid content per gram dry weight was the same in all the germ layers. Total phospholipids were analyzed quantitatively by thin layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and inositol phospholipid constituted 34, 13, 12, and 34 per cent, respectively, of the total lipid phosphorus. The phospholipid composition was different in each germ layer. The possible role of specific lipids in embryonic induction and differentiation is discussed.     

Analysis of gene expression in non-regressed and regressed bovine corpus luteum tissue using a customized ovarian cDNA array

The lifespan of the bovine corpus luteum (CL) is an important factor in the control of normal ovarian cyclicity and the establishment and maintenance of pregnancy. There is increasing evidence that CL lifespan is regulated by alternative expression of genes that promote or inhibit luteolysis. To gain further insights into these events a 434 character ovarian cDNA array comprising genes attributed to key aspects of CL function including more than 100 anonymous expressed sequence tags (ESTs) was constructed and screened with alpha(33)P dATP labeled RNA isolated from non-regressed (n=6) and regressed (n=6) CL tissue. Significance analysis of microarrays (SAM) identified 15 genes that changed expression 1.7-fold or more with a false discovery rate of <5%. The differentially expressed genes encoded enzymes involved in steroid biosynthesis and oxygen radical metabolism and proteins involved in extracellular matrix remodeling, apoptosis and cell structure. Results for five of the differentially expressed genes including matrix gla protein and collagen alpha1(I) (extracellular matrix), glutathione-S-transferase alpha I (oxygen metabolism), clusterin (apoptosis) and scavenger receptor BI (steroid biosynthesis) were confirmed by Northern blot analysis and found to be significantly different (P<0.01) between non-regressed and regressed CL tissue. Collectively this study identified genes with recognized roles in CL regression, genes with potential roles in this process and genes whose function have yet to be defined in this event.     

Changes in patterns of protein synthesis during haustorial development of Striga hermonthica (Del.) Benth. seedlings

This study focuses upon the developmental transition of theparasitic plant Striga hermonthica from its freeliving state(germinated seedling) to its parasitic state after developmentof an infection organ: the haustorium. A new method has beendeveloped that allows the production of gram quantities of germinatedand haustorially-induced Striga seedlings, thereby facilitatingbiochemical and molecular analysis of haustorial induction.Water-soluble proteins have been extracted from germinated seeds(stage A) and seedlings treated with 2,6-dimethoxy-p-benzoquinone(2,6-DMBQ) to induce haustorium (stage B). Samples were analysedby two-dimensional polyacrylamide gel electrophoresis and quantitativeas well as qualitative differences could be observed. In particulara group of four highly abundant acidic proteins (molecular weight39 kDa, pl 5.1, 5.3, 5.3, 5.6) and three other proteins (molecularweight 12 kDa, pl 6.9; 17 kDa, pl 4.4; 17 kDa, pl 4.45) wereseen in stage A while at least four proteins (molecular weight21.5 kDa, pl 6.4; 21.5 kDa, pl 6.3; 31 kDa, pl 5.1; 34 kDa,pl 6.2) were present in greater abundance in stage B. In orderto compare watersoluble protein with newly synthesized proteinpatterns, mRNAs from the two stages of development were isolatedand cell-free translation products analysed by 2-D PAGE. Two-Dgels of cell-free translation products showed the appearanceof six proteins in stage B (molecular weight ranging from 10to 35 kDa) and the presence of three acidic proteins in stageA with one protein (molecular weight 40 kDa) very similar insize to the triplet of proteins in the water-soluble protein2-D gels. Key words: Striga hermonthica (Del.) Benth., haustorium, 2-D PAGE, 2,6-DMBQ, translation in vitro     

Identification of three proteins that associate in vitro with the Leishmania (Leishmania) amazonensis G-rich telomeric strand.

The chromosomal ends of Leishmania (Leishmania) amazonensis contain conserved 5'-TTAGGG-3' telomeric repeats. Protein complexes that associate in vitro with these DNA sequences, Leishmania amazonensis G-strand telomeric protein (LaGT1-3), were identified and characterized by electrophoretic mobility shift assays and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form (a) with double-stranded DNA and the C-rich telomeric strand, (b) in competition assays using specific telomeric DNA oligonucleotides, or (c) after pretreatment with proteinase K. LaGT1 was the most specific and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a double-stranded DNA bearing a 3' G-overhang tail. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified, after renaturation, as approximately 35 and approximately 52 kDa bands, respectively. The 相似文献   

Bovine corpus luteum is an extrapituitary site of prolactin production

Prolactin (PRL) is known to be synthesized not only in the anterior pituitary, but also in other organs including the ovary. Among its various functions, PRL is regarded as the most important constituent of the luteotropic complex in rodents and pigs. The purpose of the present study was to determine whether PRL is produced locally in bovine corpus luteum (CL) and to determine its possible roles in CL. In the present study, we examined changes during the luteal phase in (1) the expressions of PRL and PRL receptors (long form: l-PRLR, short form: s-PRLR) in CL and (2) the localization of PRL in CL. We also measured the levels of PRL mRNA in cultured luteal cells and luteal endothelial cells. Furthermore, the effect of PRL on progesterone (P4) and prostaglandin (PG) F2alpha production by cultured bovine luteal cells was examined. Semiquantitative RT-PCR analysis revealed that the mRNAs for PRL and its two receptors, l- and s-PRLR, were expressed in all luteal stages examined. PRL mRNA expression was less in the regressed stage (days 19-21 after ovulation) than in the other stages. Both l-PRLR and s-PRLR mRNA expressions were higher in the late luteal stage (days 15-17) than in the other stages, while the ratio of l-PRLR to s-PRLR was less in the regressed stage than in the other stages. PRL mRNA was also detected in cultured luteal cells and luteal endothelial cells. PRL protein was immunohistochemically detected only in CL of the mid- and regressed stages. It was detected in smooth muscle cells of the intraluteal arterioles and endothelial cells but not in luteal cells and other cell types of CL. Exposure of cultured luteal cells obtained from mid-stage CL (days 8-12) to bovine PRL (100, 200 ng/ml) for 24 hr did not affect P4 and PGF2alpha production by the cells. The present study demonstrates for the first time the expressions of PRL and PRLR mRNA in bovine CL throughout the luteal phase. The overall results strongly suggest that the bovine CL is an extrapituitary site of PRL production.     

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. Mol. Reprod. Dev. 77:944–953, 2010. © 2010 Wiley‐Liss, Inc.