In vitro and in vivo association of an oviduct estrus-associated protein with bovine zona pellucida

The interaction between the bovine egg zona pellucida and a 97 kDa estrus-associated protein produced by the oviduct was examined in vitro and in vivo. In vitro matured bovine eggs were incubated with oviduct fluid recovered throughout the estrous cycle from separate indwelling cannulae placed in the ampulla and isthmus of the same oviduct. Immunofluorescence techniques and a polyclonal antiserum against the 97 kDa protein were used to localize this protein on washed eggs previously incubated with oviduct fluid. Intensity and distribution of immunofluorescence varied with stage of cycle and to a lesser degree with region of oviduct from which the oviduct fluid was obtained. The most intense fluorescence was observed on the zonae pellucidae of eggs incubated with oviduct fluid pooled from days near estrus and ovulation compared to fluid pooled from luteal stage days. The immunofluorescence of isthmus-derived oviduct fluid was more intense than was ampulla-derived oviduct fluid collected near estrus. The zonae pellucidae of 7-day-old embryos flushed from the uterus displayed immunofluorescence comparable to that observed on the zonae pellucidae of eggs incubated in vitro with peri-estrus oviduct fluid. No immunofluorescence was observed associated with the perivitelline space, egg cytoplasm, or blastomeres. The apparent uptake of a 97 kDa estrus-associated protein by the zonae pellucidae of eggs in vitro and embryos in vivo may indicate that this protein functions in fertilization and/or early embryo development.     

Secretion of proteins by cultured bovine oviducts collected from estrus through early diestrus

To characterize in vitro protein secretion by the oviduct throughout early stages of the estrous cycle, 16 cows received a luteolytic dose of PGF2 alpha and were randomly assigned to be killed on day (D) 0, 2, 5, or 8 after subsequent estrus. Explant cultures of oviducts (ampulla and isthmus) were incubated for 24 h at 39 degrees C in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5-3H]leucine. Oviductal secretion of de novo synthesized protein measured by incorporation of [3H]leucine into nondialyzable radioactivity in culture supernatants was greatest at D 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable macromolecules in tissue homogenates was also greatest at D 0. Analysis of culture supernatants by one-dimensional polyacrylamide gel electrophoresis revealed four major bands of radiolabeled proteins at greater than 97 kDa, 85-97 kDa, 55 kDa, and 30 kDa. Analysis of individual polypeptides resolved by two-dimensional polyacrylamide gel electrophoresis indicated that five of 32 individual polypeptides examined were secreted at significantly greater rates at estrus than at other times examined. One of these five polypeptides, a 97-kDa peptide with an apparent pI of 5.0, was the major secretory product at estrus and accounted for 18% of total radioactivity recovered from two-dimensional gels. Two of 32 polypeptides examined were secreted at significantly greater rates by explants of the oviduct contralateral to the side of ovulation. In summary, estrus is associated with an elevation in total protein secretion by the bovine oviduct. This increase is due to selective amplification of secretion of several but not all secretory proteins.     

Identification,characterization and localization of three proteins expressed by the porcine oviduct

At estrus, the oviduct undergoes endocrine-induced changes which provide an essential microenvironment for maturation of gametes, fertilization and embryonic development. Several oviduct expressed proteins which interact with gametes or embryos, including the oviduct-specific, estrogen-dependent glycoprotein (OGP), have been identified and characterized. The objective of the present study was to identify, characterize and localize other proteins expressed by the porcine oviduct during estrus that may function in an autocrine or paracrine manner to enhance fertilization and embryonic development. Oviducts were collected during the estrous cycle or early pregnancy, flushed and divided into functional segments, and portions of the infundibulum, ampulla and isthmus were fixed for immunocytochemical analysis or cultured. Culture media was semi-purified by heparin-agarose affinity chromatography, proteins were transferred to polyvinylidene fluoride (PVDF) membrane after two-dimensional (2D)-SDS-PAGE and three different proteins were identified, excised and subjected to N-terminal amino acid analysis. These proteins were identified as complement component C3b, the carboxy-terminal propeptide of alpha 1 (III) procollagen (PIIICP), and the heavy chain variable region of IgA. Electrophoresis and fluorography of media from Days 0 to 12 of early pregnancy or the estrous cycle revealed both spatial and temporal expression of C3b and IgA heavy chain but not PIIICP by the oviduct. Further, all three proteins were identified in oviduct fluid by electrophoresis, immunoblot or immunoprecipitation analysis. Complement component C3b and IgA heavy chain were immunolocalized in all three oviduct segments on all days; however, temporal and spatial differences were demonstrated. Staining was greater in the infundibulum and during estrus for all three identified proteins. In summary, three proteins expressed by the oviduct at estrus and during early pregnancy were identified; characterization and localization suggest they may play a critical role in protecting the luminal environment, participating in ECM remodeling and gamete interactions.     

Capacitation of bovine spermatozoa by oviduct fluid

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.     

GRP78 expression and immunohistochemical localization in the female reproductive tract of mice

The 78-kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone, with multiple functional roles in protein processing and provision of cellular protection. However, the physiological role of GRP78 in embryo development is not clear. Localization of GRP78 and expression of its mRNA in the reproductive organs throughout the estrous cycle in mice were investigated by immunohistochemistry and real-time polymerase chain reaction, respectively. Whereas there was intense staining for GRP78 in the oviduct at estrus, the ciliated cells of isthmus had better staining than those of infundibulum and ampulla at all phases of the cycle. Furthermore, GRP78 was located in the uterine luminal and glandular epithelial cells throughout the estrous cycle, particularly during the estrus phase. However, levels of GRP78 mRNA in the oviduct and uterus varied during the cycle, with peaks at estrus. In conclusion, GRP78 expression varied with the phase of the murine estrous cycle; this might be related to gamete transport, fertilization and early development of the zygote/embryo.     

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.     

Oviduct-specific expression of tissue plasminogen activator in laying hens

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

Cannulation of the equine oviduct and chemical analysis of oviduct fluid

Siliconized rubber tubes were used to cannulate one oviduct in 7 mares, and secretions were collected in a polycarbonate container located externally, in the region of the left paralumbar fossa. Secretion rates were recorded daily during the estrous cycle. Concentrations of calcium, magnesium, sodium, potassium, inorganic phosphorus and glucose were determined in the oviduct fluids secreted throughout the estrous cycle. Secretion rates were greatest during estrus (days 1-9), with a significant decrease (P<.01) noted during nonestrus (days 10-21). Concentrations of all constitutents measured were low during estrus, with a marked elevation in concentration during the nonestrus period. Histologic examination of oviducts following long-term cannulation demonstrated infiltration of lymphocytes, plasma cells, and small areas of degenerated epithelium.     

Sociosexual behavior and the ovulatory cycle of ponies (Equus caballus) observed in harem groups

Observations of sociosexual behavior of adult ponies, made on two harem groups (each comprised of one vasectomized male and three females), were correlated with follicular development and ovulation for a total of 15 cycles (minimum of 2 cycles per female). Mean cycle length (interovulatory interval) was found to be 19.7 days, with behavioral estrus lasting 7–8 days (5.5 days preovulatory; 2.3 days postovulatory). Estrous females typically showed increased frequencies of approaching and following the stallion, urinating, presenting, clitoral winking, and tail raising. Approaching and following the stallion appeared earlier and persisted longer than other estrous responses. Deviations from the modal estrous pattern included cycles with subestrus, continual estrus, behavioral estrus in the absence of ovulation, and displays of female mounting. Dominance tests revealed that a mare's status was unaffected by the phases of the estrous cycle. The presence of more than one estrous female affected the copulatory performance of both stallions, most notably in reduced latencies to first mount, intromission, and ejaculation, in spite of differences between the stallions in sexual vigor. Each stallion usually selected the dominant mare for copulation when there were multiple estrous females present, but mounts were not displayed exclusively to one female per test. The social testing situation made apparent the importance of use of space in sociosexual communication in this species, particularly in avoidance of the stallion by diestrous mares and standing alone or in proximity to him by estrous mares.     

Serum estradiol-17beta concentrations during spontaneous silent estrus and after prostaglandin treatment in the mare

Serum estradiol-17beta concentrations were determined during silent estrus in the mare. Relationships between serum estradiol-17beta concentration, corpus luteum regression, follicular development, ovulation, prostaglandin treatment and behavioral estrus were investigated. The expression of behavioral estrus was found to be related to the patterns of progesterone and estradiol-17beta secretion during the periovulatory period. When compared to normal estrous cycles, silent estrus was accompanied by a significantly lower maximum serum estradiol-17beta concentration (47.8 vs 34.6 pg/ml), a significantly longer interval from maximum estradiol-17beta concentration to ovulation (1.7 vs 4.0 days), and a significantly shorter interval from corpus luteum regression to ovulation (5.3 vs 2.8 days). Silent estrus following prostaglandin treatment was related to a significantly shorter interval from prostaglandin treatment to ovulation (3.6 +/- 0.4 days) than from normal corpus luteum regression to ovulation (5.3 +/- 0.3 days). Silent estrus appeared to be related to changes in follicular estradiol-17beta secretion and to the pattern of its secretion as related to regression of the corpus luteum. There appeared to be not only less estradiol-17beta present, but also less time available after luteal regression for it to interact with the central nervous system to elicit the changes necessary to cause behavioral estrus. There fore, unusual relationships between luteal function and folliculogenesis can result in one type of silent estrus. Significant correlations (P<0.05) were found between follicle size and serum estradiol-17beta concentration whenever behavioral estrus occurred [follicle diameter in mm = 0.96 (serum estradiol-17beta in pg/ml) + 6.08 and 0.73 (serum estradiol-17beta + 13.32 for control and normal estrus following prostaglandin treatment groups, respectively]. During silent estrus, however, no significant correlations between follicle size and serum estradiol-17beta concentration were observed.     

Luteal competency during the resumption of ovarian cyclicity in postpartum Brahman cows

Twenty pluriparous, spring-calving Brahman cows were used to determine luteal competency, as measured by serum progesterone concentrations, during the first and the second postpartum estrous cycles. Prior to and after calving, all cows were maintained in good body condition on Coastal bermudagrass pasture (IFN 1-00-703). The calves were allowed to suckle ad libitum, and sterile marker bulls were maintained with the cow herd as an aid in estrus detection throughout the trial. Cow weight and body condition score were recorded within 24 hours after calving and again at the first behavioral estrus observed. From day 1 through day 14 (day 0 = estrus) of both the first and the second postpartum estrous cycles, blood samples were collected from each cow, processed to yield serum and analyzed by radioimmunoassay for progesterone concentrations. There was a higher incidence of abnormal estrous cycles following the first postpartum estrus (35%) than following the second (5%) postpartum estrus (P<0.05). The abnormal first estrous cycles were characterized by either a short luteal phase (four cows) or by standing estrus behavior without luteal tissue formation (three cows). When serum progesterone concentrations were compared for all cows during the first estrous cycle with those during the second estrous cycle, there was less progesterone released during the cycle (P<0.05) and lower peak progesterone concentrations (P<0.10) during the first estrous cycle. However, if the abnormal cows were excluded from the analyses, there was no difference (P>0.10) in either progesterone concentrations through the 14 days measured or in peak progesterone concentrations between the first and the second postpartum estrous cycles. It can be concluded from this study that the higher incidence of abnormal luteal function following the first postpartum estrus may contribute to the decreased conception rates observed when cows are bred at their first postpartum estrus.     

Evidence of ovulation in goats (Capra hircus ) with short estrous cycle and its occurrence in the tropics

The influence of season and exposure to buck on the length of the estrous cycle were determined in 32 normally cycling native goats of different ages and parity. The estrous cycle durations were classified as short, medium and long. Ten goats (31%) exhibited medium length estrous cycles; 22 animals (69%) exhibited a combination of short, medium and long estrous cycles. Of the 155 estrous cycles studied, 15% were short, 72% were medium and 13% were long. Short estrous cycles were significantly shorter (P < 0.01) in estrus duration than medium or long estrous cycles. Significant differences (P < 0.05) for estrous cycle length and estrus duration were found between does but not within does. The presence of a buck for 8 to 16 h showed no significant effect on the length of the estrous cycle or on estrus duration, but a 24-h exposure period to a buck yielded shorter estrous cycles and estrus duration than found in the unexposed control group. Estrous cycles were significantly shorter (P < 0.01) in October, when rainfall is 57.9% of the annual total, and significantly longer (P < 0.01) in February, when rainfall is low at 0.2% of the annual total. A negative linear relationship (r = -0.87) was observed between estrous cycle and rainfall. Twenty-eight (90.3 %) of the short estrous cycles were ovulatory. The short cycles had a relatively lower ovulation rate than medium and long estrous cycles. The results indicate that the short estrous cycle in goats is associated with ovulation, and its occurrence in the tropics is related to rainfall.     

Concentrations of 13, 14-dihydro-15-keto-prostaglandin F(2)alpha, estradiol-17beta and progesterone during the peripubertal period in heifers

Twenty prepubertal Holstein heifers were utilized to assess plasma 13, 14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM), serum progesterone (P(4)) and estradiol-17beta (E(2)) concentrations as well as the E(2):P(4) ratio during the onset of puberty in cattle. All animals were maintained as a group along with a sterile marker bull to assist in the detection of estrus. Upon detection of the first estrus (Day=O), daily blood samples were collected from a jugular vein until the heifers had completed 3 estrous cycles. The average body weight and age at first estrus were 247.6+/-4.8 kg and 304.0+/-7.5 days, respectively. Frequency of abnormal length estrous cycles was greater (P<0.02) during the first (40%) and second (35%) cycles than during the third estrous cycle (0%). All heifers had normal cycle lengths (18 to 24 days) by the third estrous cycle. Serum P(4) was greater during the third cycle (P<0.05) from Day 10 to Day 4 before the next estrus compared with the same period of the first estrous cycle. Serum E(2) did not peak until the day of estrus in the first cycle, whereas E(2) reached a maximal level 2 days before estrus in the third estrous cycle. Serum E(2) was higher (P<0.0001) 2 days before estrus in the third cycle than in the first estrous cycle. Plasma PGFM reached maximum concentrations 3 days before estrus in the third cycle compared with 1 day before estrus at the end of first estrous cycle. As estrus approached during the third cycle, PGFM rose 1 day before E(2) rose and P(4) declined, while the rise in PGFM and E(2) occurred simultaneously, with P(4) declining at the end of the first estrous cycle. During diestrus, the E(2):P(4) ratio was lower (P<0.07) in the third cycle than in the first, but it was higher (P<0.04) at estrus and 1 day before in the third estrous cycle. These data reveal a high incidence of abnormal length estrous cycles during the first two estrous cycles of the peripubertal period, and demonstrate anomalies in uterine and ovarian endocrine activity during the peripubertal period in cattle.     

Lysosomal hydrolases in reproductive organs during estrous cycle of the hamster

Changes in the total protein content and the activities of lysosomal hydrolases (arylsulfatase, acid phosphatases, β-glucuronidase, β-N-acetylhexosaminidase, α-L-fucosidase, and β-galactosidase) of the hamster genital tract during the 4 days of estrous cycle and in hormonally superovulated hamsters were measured. Levels of lysosomal hydrolases in uteri and uterine fluid changed significantly during the cycle. Similar changes were observed in uterine wet weight and uterine proteins. The pattern of enzyme activities in both the ovary and the oviduct were different from those in uteri. In the ovary, most enzyme activities and the total protein concentration remained elevated after ovulation. Protein concentration and enzyme activities were significantly higher in the ovary, oviduct, and uteri of superovulated hamsters as compared to controls.     

Changes in the composition of cervical mucus of the cow during the estrous cycle as parameters for predicting potential fertility

Four experiments were conducted to test the hypothesis that the composition of cervical mucus can be used as an indicator of reproductive efficiency in the cow. In Experiment 1, biochemical changes were studied in cervical mucus during the estrous cycle. Sorbitol concentration was observed to be highest at 1 to 3 days prior to estrus and lowest on Days 6 to 12 (P<0.001) of the estrous cycle. Cholesterol and protein concentrations were highest at Day 6 of the estrous cycle and lowest on the day of estrus (P<0.001). In Experiment 2, the relationships between the biochemical characteristics of cervical mucus and fertility were studied. It was shown that the embryo transfer recipients which exhibited a high concentration of sorbitol (>1.5 mMol/l) at 1 to 3 days before estrus; a low concentration of protein (< 2 units); and a low concentration of cholesterol (<0.1 mMol/l) on the day of estrus had a higher level of fertility than their counterparts. The predictive ability of these criteria was tested using embryo transfer recipients (n=294) in Experiment 3. Significantly more of the animals predicted to have high potential fertility became pregnant than those predicted to have low potential fertility (70.7 vs 45.6%; P<0.001). A similar difference in pregnancy rate for cows (n=56) presented for artificial insemination was observed in Experiment 4 (59.1 vs 27.2%; P<0.10). These results suggest that the composition of cervical mucus may be a useful indication of potential fertility in cattle.