Estrous Cycle In Cattle Research Articles

Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro.

Spontaneous contraction of oviductal smooth muscle is essential for gamete transport to the fertilization site in mammals. This study sheds light on the mechanism of elevated contraction amplitude in the bovine oviductal isthmus just before ovulation. Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0-4 days after ovulation), at the mid-late luteal stage (10-17 days), and at the follicular stage (1-3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher in the follicular stage. In addition, estradiol-17β (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

Metabolomic Profile of Oviductal Extracellular Vesicles across the Estrous Cycle in Cattle

Oviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs’ metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development.

Progestogen profiling in plasma during the estrous cycle in cattle using an LC-MS based approach

In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABAA-r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20β-dihydroprogesterone (20β-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3β-dihydroprogesterone (3β-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5β-reduced progestogens (5β-dihydroprogesterone (5β-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20β-DHP. Further studies will need to focus particularly on the period around luteolysis.

Stage-dependent changes in oviductal phospholipid profiles throughout the estrous cycle in cattle

Sperm capacitation, fertilization and embryo development take place in the oviduct during the periovulatory period of the estrous cycle. Phospholipids are crucial metabolites for sperm capacitation and early embryo development. The aim of this study was to monitor the abundance of phospholipids in the bovine oviductal fluid (OF) according to the stage of the estrous cycle and the side relative to ovulation. Pairs of bovine oviducts were collected in a slaughterhouse and classified into four stages of the estrous cycle: post-ovulatory (Post-ov), mid-luteal (Mid-lut), late-luteal (Late-lut) and pre-ovulatory (Pre-ov) phases (n = 17 cows/stage). Cell-free OF from oviducts ipsilateral and contralateral to the site of ovulation were analyzed using MALDI-TOF mass spectrometry. Lipid identification was achieved by high resolution mass spectrometry. A total of 274 lipid masses were detected in the mass range of 400–1000 Da, corresponding mostly to phosphatidylcholines (PC), lysoPC, phosphatidylethanolamine (PE), lysoPE and sphingomyelins (SM). Ipsilateral and contralateral OF did not differ in their lipid profiles at any stage of the cycle. However, 127 and 96 masses were differentially abundant between stages in ipsilateral and contralateral OF, respectively. Highest differences in lipid profiles were observed in the Pre-ov vs. Mid-lut and Pre-ov vs. Late-lut comparisons in both sides relative to ovulation. Differential abundance of specific molecules of PC, PE, SM and l-carnitine were observed at Pre-ov and Post-ov compared with the luteal phase. This work proposes new candidates potentially able to regulate sperm capacitation and early embryo development.

Physiological characteristics and effects on fertility of the first follicular wave dominant follicle in cattle

The first follicular wave emerges soon after ovulation, and its dominant follicle (DF) develops during the first 8–10 days of the estrous cycle in cattle. And, the first-wave DF is a non-ovulatory follicle, because it develops during the first half of the estrous cycle simultaneously with the corpus luteum (CL), which produces and secretes progesterone. Regarding the characteristics of development and the mechanisms of deviation in the DF during the follicular wave, the first-wave DF has been well studied. However, the characteristics of the first-wave DF, such as growth, blood flow in the follicular wall, concentration of sex steroid hormones in the peripheral blood and follicular fluid, amounts of mRNA in granulosa cells, as well as the characteristics of the CL formed after the first-wave DF and the influence of the first-wave DF on fertility (conception rate), have not been well studied. Additionally, the first-wave DF synthesizes and secretes 17β-estradiol (E2), and plasma E2 concentration increases during the early stage of the estrous cycle. Consequently, there is a possibility that the first-wave DF might affect the fertility in cattle. In this review, to provide the new perspective on reproductive physiology in cattle, characteristics of the first-wave DF were examined in detail and its characteristics were compared with that of the second-wave DF. In addition, the locational effects of the first-wave DF and CL on conception rate are discussed.

Effect of Kisspeptin-10 on plasma luteinizing hormone concentrations and follicular dynamics during the luteal phase in cattle

Two experiments were carried out to determine the effect of Kisspeptin-10 on plasma LH concentrations and follicular dynamics during the luteal phase in cattle. We tested the hypothesis that a single treatment of Kisspeptin-10 will increase plasma LH concentration and the diameter of the dominant follicle, and induce ovulation during the luteal phase of the estrous cycle in cattle. In the Experiment 1, Hereford-cross heifers (n = 28, 14–16 mo) were given PGF2α im to induce luteolysis and ovulation. On Day 5 (Day 0 = ovulation), a new follicular wave was induced by ultrasound-guided follicular ablation. Heifers were treated on Day 10 (4 days after wave emergence) with 100 μg GnRH im (n = 9), 2 mL saline im (n = 7), 1 mg Kisspeptin-10 im (Kp im, n = 6) or 1 mg Kisspeptin-10 iv (Kp iv; n = 6). Blood samples were collected at −60, −15, 0, 5, 15min (0 min = time of injection) and every 15 min thereafter until 3 h. Transrectal ovarian ultrasonography was performed at 12 h intervals from Day 10–14. In Experiment 2, non-lactating beef cows on Day 5 were treated with 100 μg GnRH im (n = 9), saline im (n = 5), 10 mg of Kisspeptin-10 iv (Kp 10 mg; n = 5) or 15 mg of Kisspeptin-10 iv (Kp 15 mg; n = 5). Blood samples were collected at −15, 0, 15, 30, 60, 120, 180 min and twice daily ovarian ultrasonography was done from Day 5–10. In Expt 1, plasma LH concentrations increased for 1 h following Kp iv administration. The peak concentration occurred at 15 min and was higher in the Kp iv group than in the Kp im group (P = 0.01). The LH peak was 3.5-folds higher in the GnRH group than the Kp iv group (P < 0.0001). In Expt 2, GnRH induced higher (P < 0.001) plasma LH concentrations for all time-points than other groups. Kp 15 mg at peak (15min), 30 and 60 min induced higher (P < 0.0001) plasma LH concentrations than Kp 10 mg and saline. Kisspeptin-treated animals did not ovulate in either experiment while GnRH induced ovulation (n = 5/9 in Expt 1; 9/9 in Expt 2). The diameter of the dominant follicle was greater (P = 0.02) at 12–48 h after kisspeptin treatment (Kp groups combined) than the Saline group in Expt 2. In conclusion, Kisspeptin-10 increased plasma LH concentrations and follicle size, and although plasma LH concentrations were higher after iv than im administration, but at the doses used, Kisspeptin-10 did not induce ovulation during the luteal phase in cattle.

Ghrelin suppresses the GnRH-induced preovulatory gonadotropin surge in dairy heifers

Ghrelin, a known growth hormone (GH) secretagogue, alters gonadotropin secretion in many species. Our objectives were to study the effects of ghrelin, on GH, LH, FSH secretion, and on luteal function of the ensuing estrous cycle in cattle. The estrous cycles of eight heifers were synchronized with progesteron releasing intravaginal device, and ovulation was induced with GnRH. Eight animals were treated with 1.5 μg kg−1 bovine ghrelin (group Ghr, n = 4) or saline (group C, n = 4). Starting with the first ghrelin injection, 13 blood samples were collected over a 4-hour period for the determination of ghrelin, GH, LH, and FSH concentration. Progesterone levels were measured in samples collected every other day after estrus expression. Data were analyzed by repeated measures of ANOVA followed by Bonferroni post hoc testing and t test. In group Ghr, ghrelin concentration increased significantly 15 minutes after the first injection and remained in elevated levels until the 90th minute after the last injection. At the time of third ghrelin injection, GH was significantly higher in the Ghr group compared with C (17.1 ± 1.3 vs. 2.6 ± 0.3 ng mL−1, P < 0.0001). Similar differences were found for the next three samples collected 15, 30, and 60 minutes later; no difference was evident after 90 minutes. In group Ghr, the area under the curve for LH and FSH were significantly reduced compared with the ones of group C (266 ± 10.3 vs. 331.9 ± 7.3, P = 0.007 and 102.3 ± 2.0 vs. 134.9 ± 5.5, P < 0.005 for LH and FSH respectively). At particular time points the concentration of the two gonadotrophins in group Ghr was significantly lower than those of group C (15, 30, 45, 75, and 90 and 60, 75, 90, 120, and 150 minutes after GnRH administration for LH and FSH respectively). The duration of the following estrous cycle was shorter (P = 0.004) in group Ghr (19.0 ± 0.4 days) compared with C (21.8 ± 0.5 days). In days 4, 6, 8, 10, and 14, progesterone concentration was lower (P < 0.05) in group Ghr compared with C; similarly the progesterone area under the curve for group Ghr (113.1 ± 4.8) was suppressed (P = 0.007) compared with that of C (141 ± 4.8). These results imply that ghrelin acts on pituitary causing impaired response to the GnRH stimulus, and it is likely to affect luteinization of the cellular compartment of the preovulatory follicle, and/or to suppress steroidogenetic activity of the luteal cells.

Characterization of follicle and CL development in beef heifers using high resolution three-dimensional ultrasonography

The aim was to characterize dominant follicle (DF) and CL development through the estrous cycle of cattle using three-dimensional (3D) ultrasonography while making a comparison with conventional two-dimensional (2D) B-mode ultrasound (US) and to relate the measures taken to systemic concentrations of steroid hormones and gonadotropins. After synchronization of estrus, the ovaries of crossbred beef heifers (N = 5) were assessed using daily US with a GE Voluson i US scanner until the end of the first follicle wave, then every other day until emergence of the final (ovulatory) wave, when daily US resumed until ovulation. Follicle and CL growth were recorded and mapped. Measures of diameter (2D) and volume (3D) of the DF from the first and ovulatory waves of the cycles; and CL development were captured and stored for further analysis. Blood flow to the DF and CL were assessed using 3D power Doppler US measuring vascularization index (VI; %), vascularization flow index (0/100) and flow index (0/100). Jugular blood samples were collected every 24 hours for progesterone from the first estrus until the second ovulation. Concentrations of estradiol (E2) and follicle stimulating hormone (FSH) were measured every 8 hours from estrus to second follicle wave emergence; then, E2 only was measured from final follicle wave emergence until ovulation. Data were analyzed using PROC MIXED and PROC REG in SAS. Dominant follicle blood flow tended to decrease during follicle wave emergence and DF VI increased (P < 0.05) 24 hours before ovulation after peak E2. Measures of the DF and CL volume (3D) were highly predictive of 2D diameter measures throughout the cycle (P < 0.0001). Predictive values (r2) for day of wave emergence and day from ovulation were similar for 2D and 3D measures; however, 2D measures had higher repeatability when compared with 3D measures. There was no relationship between CL VI and progesterone early in the cycle (r2 = 0.12; P = 0.1); however, there was a strong positive relationship approaching ovulation (r2 = 0.77; P < 0.0001). In conclusion, 3D power Doppler measures of blood flow appears to be representative of vascular changes in the DF and CL throughout the estrous cycle. However, the extra time required to acquire and analyze a 3D image and the relatively little additional information obtained over that achievable with 2D imaging in terms of follicle and CL development might preclude its widespread use other than for detailed research purposes.

Presence of a Temperature Gradient Among Genital Tract Portions and the Thermal Changes Within These Portions Over the Estrous Cycle in Beef Cows

The aim of the present study was to describe the temperature of the different portions of the female genital tract and their relation to rectal temperature and to investigate the effect of steroid hormones profiles on these variables over the estrous cycle in cattle. Four nonpregnant Japanese Black cows were investigated daily over two successive estrous cycles using a digital thermometer with a long probe and rounded-end sensor to record the temperature of the rectum (RT), vagina (VT), cervix (CT), uterine body (UBT) and uterine horns (UHT). Blood samples were collected immediately before temperature recording to assay peripheral levels of progesterone (P4) and estradiol-17β (E2). Moreover, transrectal ultrasonography was carried out after temperature recording to monitor the ovulatory follicle and track ovulation. During the experiment, the ambient temperature and relative humidity were recorded for further calculation of the temperature humidity index (THI). The temperature within the genital tracts in these cows progressively increased towards the uterine horns from the vagina. The VT, CT, UBT and UHTs were significantly higher in association with peripheral P4 concentrations greater than 4 ng/ml (mid-luteal phase) when compared with lower peripheral P4 concentrations. The VT was more significantly (P<0.01) correlated to the CT, UBT and UHTs than RT. In conclusion, a temperature gradient was present among the vagina, cervix and uterus over the estrous cycle, and changes in peripheral P4 concentrations were associated with the thermal variations within these portions. The VT could be more beneficial than RT in monitoring temperature of deeper portions of the female genital tract in bovine.

Proteomic Characterization of Histotroph during the Preimplantation Phase of the Estrous Cycle in Cattle

Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.

ANIMAL MODELS FOR THE STUDY OF HUMAN OVARIAN FUNCTION

The study of ovarian function has been of great interest to scientists and clinicians in the human and veterinary health fields for more than 20 centuries. Initial studies of the ovaries were based on anatomical descriptions post-mortem, followed by histological and endocrinological approaches. The introduction of high-resolution ultrasonography provided a long-awaited tool to image the reproductive tissues in situ in both animals and humans. Critical studies of the characteristics and control of ovarian follicular and luteal dynamics in non-human primates, rodents, and domestic farm animals have involved frequent (i.e. daily or multiple times a day) blood sampling and ultrasonography. Studies of this nature in women are difficult, and often unethical to conduct. Differences in antral folliculogenesis between humans and animals appear to be more in detail rather than in essence, and may reflect differences in intrinsic physiology or merely differences in our ability to detect changes in a given species. In women, the presence of endometrial shedding and symmetric luteal and follicular phases are different from that observed during the estrous cycles of domestic farm animals but despite these differences, general similarities in antral follicular dynamics exist. A continuous pattern of antral follicle development was originally proposed in domestic livestock species; however, the use of frequent serial ultrasonography and simultaneous endocrine profiling in these animal species has resulted in a broad understanding of follicular wave dynamics. Follicular waves have now been described in every species in which this approach has been used, including humans. The relatively large diameters of antral follicles in cows and mares, compared to monkeys, sheep, and rodents provide greater feasibility for characterizing antral follicular dynamics ultrasonographically. Profound similarities in the dynamics of follicle development exist between the menstrual cycle in humans and the estrous cycle in cattle and horses. Despite a proportionately longer luteal phase in cows and mares, the growing phase of the dominant ovulatory follicle (defined as the time period from wave emergence to ovulation) is comparable in the human, cow, and mare. Thus, at present, cows and mares may be the most appropriate models for the study of antral follicular dynamics in women. While the use of animal models has increased our understanding of ovarian function and provides the hypothetical basis for studies in women, differences in vocabulary, culture, and research methodologies between scientists in predominantly human versus animal settings has hampered knowledge translation. These differences represent a systemic impediment to a broad understanding of ovarian function and limits progress and innovation in the development of safer and more efficacious treatments for infertility and contraception.

The ability of subordinate follicles of the second follicular wave to become dominant is lost by day 15 of the estrous cycle in cattle

Generally, unilateral ovariectomy before a critical period in the latter part of the estrous cycle induces a transitory increase in plasma FSH, which causes subordinate follicles to develop and maintain ovulation rates characteristic of the species. A limiting period for subordinate follicles to assume dominance and from which ovulation occurs has not been shown for cattle. Growth and/or regression of subordinate follicles were characterized following removal of the dominant follicle at different days of the luteal phase of the estrous cycle in cattle in this study. In the mid-luteal phase (Day 13 or 15), the ovary with the dominant follicle of the second wave was ablated via unilateral ovariectomy; the corpus luteum also was removed. In the late luteal phase (Day 17 or 19), the dominant follicle was ablated with an ultrasonically guided 20 gauge needle. When the dominant follicle was removed on Day 13, the largest subordinate follicle of the second wave of follicular development became dominant and ovulation occurred from this follicle in 4 of 4 animals. However, when the dominant follicle was removed on Day 15, 17 or 19, a new wave of follicular development was induced in 14 of 15 animals. Moreover, the recovered subordinate follicle of the second wave of follicular development had similar growth characteristics to naturally occurring dominant follicles. In conclusion, the subordinate follicle in the second follicular wave in cattle retained the ability to become dominant, but this ability was lost by Day 15 of the estrous cycle. However, cattle then were able to maintain ovulation by developing a new wave of follicular growth.

Effect of medroxy-progesterone acetate on follicular growth and endometrial cycloxygenase-2 (COX-2) expression during the bovine estrous cycle

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P&lt;0.05) in all cows except controls and those receiving 250mg MAP without EB. Seventy-five percent of the animals (15/20) showed estrus on days 21 and 22 of the cycle rather than at MAP withdrawal, demonstrating that these treatments did not induce estrus. To determine whether the EB treatment altered endometrial sensitivity to oxytocin and thus the luteolytic cascade, multiparous pre-synchronized cows received 5 mg of EB followed 6 hours later with 50 IU of oxytocin (OT; n=9). Eight hours after EB injection, endometrial fragments were collected from the cows on days 4, 13 and 17 of the estrous cycle and COX-2 gene expression was measured by PCR. EB increased COX-2 mRNA levels only on day 17 of the estrous cycle (P&lt;0.05). In conclusion, MAP alone or associated with EB is able to suppress bovine follicular growth. However, EB in the presence of MAP is not efficient to induce luteolysis in cows when injected on day 8 of the estrous cycle.

Relationship between concentrations of progesterone, oxytocin, noradrenaline, gene expression and protein level for their receptors in corpus luteum during estrous cycle in the cow

The aim of these studies was to evaluate of the relationship between luteal concentrations of oxytocin (OT), noradrenaline (NA), progesterone (P4), oxytocin receptors (OT-R) and β 2-adrenoreceptors (β 2-R) gene expression and their protein level throughout the estrous cycle in cattle. Corpora lutea (CL) collected during days 1–5, 6–10, 11–16 and 17–21 of the estrous cycle were used in these studies. Concentrations of P4, OT and NA were determined in tissue extracts. Gene expression and protein level for OT-R and β 2-R were investigated by RT-PCR and Western blot, respectively. Luteal concentration of P4 was higher ( P < 0.01) on days 6–10 and 11–16 than during days 1–5 and 17–20 of the estrous cycle. Concentration of OT was the highest on days 1–5 and 6–10 of the estrous cycle. This concentration decreased ( P < 0.01) during days 11–16 reaching the lowest level ( P < 0.001) on days 17–20 as opposed to days 1–10 of the estrous cycle. Expression of OT-R mRNA was lower on days 6–16 ( P < 0.05) followed by its increase on days 17–20 as opposed to the expression observed on days 1–5. Expression of OT-R mRNA was negatively correlated ( P < 0.05) with the profile of OT-R protein level. This latter parameter was the lowest on days 17–20 and 1–5, and the highest on days 6–10 and 11–16. Oxytocin concentration was negatively correlated ( P < 0.05) with expression of OT-R mRNA and positively correlated ( P < 0.001) with OT-R protein level. This protein level was only correlated with P4 ( r = 0.59; P < 0.05). Concentration of OT was positively correlated with level of P4 ( P < 0.001). Concentration of NA was the highest on days 1–5 of the estrous cycle, whereas it was similar or lower ( P < 0.05) on days 6–21. Expression of mRNA for β 2-R was the lowest on days 1–5 and was highly increased ( P < 0.05) on days 6–16. The expression was the highest ( P < 0.001) on days 17–21 compared to all others group of the estrous cycle. Protein level for β 2-R was the highest on days 1–5 and decreased ( P < 0.05) on days 6–10 and 11–16. The expression was the lowest on days 17–20 compared to the β 2-R protein level in CL from all others stages of the estrous cycle. Protein level for β 2-R was positively correlated ( P < 0.05) with the OT concentration. Expression of mRNA for β 2-R was negatively correlated ( P < 0.001) with level of β 2-R protein. No correlation was found between β 2-R mRNA expression and NA concentration or between NA and P4 concentrations. Results presented in this study suggest an evident relationship between OT and NA and may play an important role in the regulation of luteal steroidogenesis during all stages of the estrous cycle.

The expression of the nuclear receptors NR5A1 and NR5A2 and transcription factor GATA6 correlates with steroidogenic gene expression in the bovine corpus luteum

The corpus luteum (CL) is the major site of progesterone (P4) production during the luteal phase of the estrous cycle in cattle. To better understand the molecular mechanisms underlying P4 production, we compared the mRNA and protein expression profiles of key components of the steroidogenic pathway (StAR, CYP11A, and 3beta-HSD) during the bovine CL luteal phase with that of several transcription factors (NR5A1, NR5A2, GATA4, GATA6) known for their roles in the control of steroidogenic gene expression. In the bovine CL, StAR, CYP11A, and 3beta-HSD mRNA and protein levels remained constant at the mid and late luteal phases but markedly declined at the regressed luteal stage. NR5A1 and NR5A2 exhibited a similar pattern with a significant decrease in expression at the regressed luteal stage. Both GATA4 and GATA6 mRNA and proteins could be detected in bovine CL; GATA6 levels, however, were generally higher. Although GATA4 expression did not change during the luteal phase, GATA6 showed a marked decrease at the regressed luteal stage, like NR5A1, NR5A2, and the other steroidogenic markers. Thus, we suggest that NR5A1, NR5A2, and GATA6, but not GATA4, contribute to the transcriptional regulation of steroidogenic gene expression, and hence P4 production, in the bovine CL. Furthermore, we have demonstrated the association of NR5A1 and NR5A2 with the bovine StAR promoter in the mid-luteal CL using chromatin immunoprecipitation, suggesting that these factors have definitive roles in the regulation of StAR gene transcription in vivo.

The adverse effect of dichlorodiphenyltrichloroethane (DDT) and its metabolite (DDE) on the secretion of prostaglandins and oxytocin in bovine cultured ovarian and endometrial cells

The influence of chlorinated hydrocarbon insecticide—dichlorodiphenyltrichloroethane (DDT) and its metabolite (DDE) on the uterine prostaglandin (PGF2α and PGE2) and ovarian oxytocin (OT) secretion was investigated. Endometrial, granulosa and luteal cells from cows and heifers on 8–12 days of estrous cycle were treated (24–72 h) with DDT, mixture or individual isomer (p,p′-, o,p′-) of DDE (0.1, 1 or 10 ng/ml). The viability of cells and the concentrations of hormones in medium were determinted. Neither DDT nor DDE affected ( P > 0.05) the viability of cells, while they stimulated ( P < 0.05) PGF2α and reduced ( P < 0.001) PGE2 secretion from endometrial cells. Hence the ratio of PGF2α to PGE2 was markedly changed. Used pollutants also increased ( P < 0.05) OT secretion from ovarian cells. It is concluded that DDT and its metabolite impair PGF2α, PGE2 and OT secretion from uterus and ovary, respectively, and this way they can affect the course of estrous cycle in cattle.

Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers ( n = 35) were ovariectomized at 36 ( n = 7), 66 ( n = 8), 84 ( n = 12) and 108 ( n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment ( n = 6) or were treated with a progesterone-releasing intravaginal device ( n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1–F4). Mean diameter of the F1 follicle increased ( P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest ( P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower ( P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108 h, but amounts of follicular fluid IGFBP-4 were lower ( P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher ( P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.

Vascular endothelial growth factor system in the cow oviduct: A possible involvement in the regulation of oviductal motility and embryo transport

Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability enhancing factor, which shows the highest activity in the oviduct during the periovulatory period of the estrous cycle in cattle. It has also been shown that the contraction activity of oviduct is highest during the periovulatory period. The present study therefore focused on the possible involvement of VEGF in the regulation of biosynthesis and secretion of contraction-relaxation-related substances in the cow oviduct. Possible autonomous VEGF system in the oviduct as well as its endocrine control was also studied. Bovine oviductal epithelial cells (BOEC) in the second passage were cultured with VEGF (1 ng/ml) alone or with luteinizing hormone (LH; 10 ng/ml), estradiol 17-beta (E2; 1 ng/ml), and/or progesterone (P4; 1 ng/ml). The levels of prostaglandins (PGs), endothelin-1 (ET-1), and angiotensin II (Ang II) in the medium were measured using second antibody enzymeimmunoassay (EIA). The mRNA expressions for cycloxygenase-2 (Cox-2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), prepro-ET-1, endothelin converting enzyme-1 (Ece-1), angiotensin converting enzyme-1 (Ace-1), VEGF and its receptors were investigated using real-time RT-PCR. The results indicate that, (1) VEGF dose-dependently stimulated the release of prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha), and ET-1, but not Ang II. VEGF and VEGF with LH, E2, and P4 upregulated mRNA expression for biosynthesis cascade of PG, ET-1 as well as their release. However, only the combination of VEGF with LH, E2, and P4 upregulated mRNA for Ace-1 and Ang II release, but not VEGF alone. (2) Treatments of LH, with E2 and/or P4 increased the mRNA expression for VEGF, Flk-1 and Flt-1, and (3) VEGF itself downregulated the expression of mRNA for VEGF, and LH, E2, and P4 enhanced this downregulatory effect. The results of the present study provide the first evidence that (1) VEGF directly stimulates the biosynthesis and release of PGE2, PGF2alpha, and ET-1 in the bovine oviduct, (2) LH stimulates the oviductal VEGF system, and (3) VEGF downregulates the oviductal VEGF system and this downregulation was further intensified in the presence of LH. The data suggest that the preovulatory LH-surge, together with increasing E2 secretion from the Graffian follicle and basal P4 levels from the regressing corpus luteum (CL), upregulates the oviductal VEGF system, inducing the maximum oviductal production of contraction-relaxation-related substances for active oviduct contraction and rapid transport of gametes to the fertilization site. However, the oviductal VEGF elevation caused by the LH-surge, appears to downregulate the oviductal VEGF system immediately after ovulation thereby may contribute to suppress oviductal contraction to secure slow transport of the embryo to the uterus at the optimal time.

Roles of tumor necrosis factor-alpha of the estrous cycle in cattle: an in vivo study.

We have suggested in a previous in vitro study that tumor necrosis factor-alpha (TNFalpha) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNFalpha on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F2alpha [PGF2alpha] and leukotriene C4 [LTC4]) and luteotropic (PGE2) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNFalpha actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF2alpha (cloprostenol, 100 microg; n = 3) or saline with TNFalpha at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 microg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNFalpha treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNFalpha (0.1 and 1 microg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 +/- 0.9 and 18.0 +/- 0.7 days, respectively) compared with the control (22.3 +/- 0.3 days, P < 0.05). One microgram of TNFalpha increased the PGF2alpha (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNFalpha (10, 25, 50 microg) stimulated synthesis of P4 (P < 0.001) and PGE2 (P < 0.001), inhibited LTC4 secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNFalpha cause luteolysis, whereas high concentrations of TNFalpha activate corpus luteum function and prolong the estrous cycle in cattle.

Use of a small dose of estradiol benzoate during diestrus to synchronize development of the ovulatory follicle in cattle.

We tested the hypothesis that a small dose of estradiol benzoate (EB) at the midstage of the estrous cycle in cattle would synchronize the subsequent pattern of ovarian follicular development, estrus, and ovulation. Nonlactating Friesian cows received either 1 mg of EB i.m. on d 13 of the estrous cycle (T; n = 12; estrus = d0) or served as untreated controls (C; n = 12). Their ovaries were examined daily with transrectal ultrasonography from d 7, and blood samples were collected 0, 2, 4, 8, 24, and 48 h after treatment on d 13. Plasma concentrations of estradiol-17beta were elevated to 12 pg/mL during the initial 24 h following treatment, compared with a baseline of 1 pg/mL in untreated controls (P < .001). Progesterone concentrations in cows of the T group declined between 24 and 48 h after treatment (-3.2 +/- .5 ng/mL) compared with little change in concentrations of progesterone in cows of the C group at this time (P < .01). This difference was coincident with an earlier time to regression of the corpus luteum in cows of the T group. Disregarding treatment groups, the second dominant follicle of the estrous cycle (DF2) emerged on d 10.6 +/- .3 and was 9.4 +/- .4 mm in diameter on d 13. Further growth of the DF2 was halted by EB treatment on d 13. Cessation of growth occurred irrespective of whether the DF2 was in the early or late growth phase, and a new follicular wave emerged 4.5 +/- .2 d later. The dominant follicle from this wave (DF3) ovulated 5 d after emergence in most cases. During the estrous cycle of every cow in the T group, there were three waves of follicular development (3-wave), whereas the ratio of 2:3 waves of follicular development in cows of the C group was 1:3. Consequently, the interval from emergence to ovulation of the ovulatory dominant follicle in cows of the C group ranged from 3 to 11 d. The dynamics of ovarian follicular wave development during the estrous cycle can be strategically manipulated by treating with a small dose of EB to synchronize proestrous development of the ovulatory follicle.