Corpus Luteum Function Research Articles

The male-induced implantation failure (the Bruce effect) in mice: effect of exogenous progesterone on maintenance of pregnancy in male-exposed females.

The effect of exogenous progesterone given early during pregnancy in maintaining pregnancy in alien male-exposed mice was investigated. Administration of progesterone, 1 mg/female/day, on days 1 to 5 post coitum was fully effective and on day 2 to 6 post coitum was partially effective in maintaining pregnancy in females. However, progesterone injection on days 3 to 7 post coitum was totally ineffective in maintaining pregnancy in females. The results suggest that only when administration of progesterone is begun early during male exposure, luteal failure in newly inseminated females is prevented. The results also suggest that failure of corpus luteum function or some other irreversible changes leading to luteolysis occur in females early during exposure to alien males before the external manifestation of implantation failure (vaginal cornification). It is suggested that exogenous progesterone given early during alien male exposure prevents luteal failure and maintains pregnancy in females through its facilitatory effect on hypophysial prolactin secretion.

Some studies on the morphological aspects of buffalo oocytes in relation to the ovarian morphology and culture condition.

The present study was conducted to establish the effects of ovarian morphology on oocyte quantity and quality, as well as the effect of preincubated granulosa cells (PGCs) on in vitro maturation of buffalo oocytes and steroid hormones production. A total of 52 ovarian pairs were grouped into three types: type I (with functional corpus luteum), type II (with regressed corpus luteum), and type III (without corpus luteum). The number of follicles and oocytes/ovary were documented. The follicles were classified into three groups (<2, 2-6, and >6 mm Ø). Oocytes were classified according to their morphology into four grades (grades A, B, C, and D), or according to their cumulus compactness into four groups (more than three layers, one to three layers of cumulus cells, partial remnants of cumulus cells, and no cumulus cells). A PGCs was used to investigate their steroidogenic potential on the in vitro maturation. The highest number of follicles and oocytes was found in type III than types II and I. Grades A and B oocytes were significantly higher (P < 0.01) in number in type III ovaries. Oocytes with more than three layers of cumulus cells showed higher maturation rate than oocytes with partial remnants or no cumulus cells but with small difference from oocytes having one to three layers of cumulus cells. Beside the higher maturation rate in compact than denuded oocytes, a significantly higher (P < 0.01) rates obtained in compact or denuded oocytes when cultured in vitro with PGCs than the corresponding oocytes with no PGCs. These maturation rates coincided with higher level (P < 0.05) of estradiol-17ss when compact oocytes cultured with or without PGCs than denuded oocytes and higher level (P < 0.05) of progesterone after culture with PGCs for both compact and denuded oocytes than the corresponding oocytes with no PGCs. In summary, buffalo ovaries with no corpus luteum may be suggested for obtaining high number of follicles and good oocytes than the others. Oocytes with intact cumulus showed better maturation than those with partial or denuded cumulus, although the denuded oocytes improved their meiotic competence to a less or greater extent when cultured in vitro with PGCs.

Effects of Interferon-Tau on Cortisol Production by the Bovine Endometrial Tissues.

Glucocorticoids (GCs) are well known as anti-inflammatory agents limiting the production of cytokines and prostaglandin (PG)s in various target organs. Access of GCs to GC receptor (GC-R) in target tissues is regulated by two 11β-hydroxysteroid dehydrogenases (11-HSD1 and 2). We previously demonstrated that cyclic bovine endometrium expresses 11-HSD1 mRNA, and converts biologically inactive cortisone into cortisol. Moreover, we have shown that cortisol suppressed PGF2α without affecting PGE2 production in the cyclic bovine endometrium. Interferon-τ (IFN-τ) produced by the trophoblast at the time of recognition of pregnancy acts on maintaining corpus luteum (CL) function. Furthermore, some previous study demonstrated that in ewes the plasma concentrations of cortisol increased in response to IFN-τ. Therefore, we suggest that cortisol acts as a luteoprotective factor by selectively suppressing luteolytic PGF2α production by the pregnant bovine endometrium as INF-τ mediator. The aim of the present study was to determine if IFN-τ affects cortisol production and secretion in bovine endometrial tissues during the early pregnancy comparing to the responsible days of the estrous cycle. In Exp. 1., GC-R, 11-HSD1 and 11-HSD2 expressions on the gene (mRNA) and protein levels in pregnant (Days 16-18 after insemination) and nonpregnant (Days 16-18 after ovulation) endometrial strips were measured by using real time RT-PCR and Western-Blot analysis, respectively. In Exp. 2. (tissue culture study), the endometrial explants were taken from cows on Days 16-18 of the cycle and pregnancy. Endometrial slices from both groups of cows were incubated for 12 h with cortisone (30 nM) and/or IFN-τ (30 ng/ml), and then 11-HSD1 activity was evaluated by measuring cortisol level. Moreover, the endometrial stromal and epithelial cells were isolated enzymaticaly from bovine uteri in the early luteal stage of estrous cycle (Exp. 3., cell culture study). The cells were seeded in 48-well cluster dishes and then were exposed to cortisone (30 nM) and/or IFN-τ (30 ng/ml). After incubation, the conditioned media were collected, and then 11-HSD1 activity was evaluated by measuring cortisol level. Endometrial GC-R and 11-HSD1 mRNA expressions were higher from pregnant than those from non-pregnant cows (P<0.05). Moreover, 11-HSD2 mRNA expression was lower in cyclic cows than that in pregnant cows. Protein expression for 11-HSD1 was significantly higher in the tissues from pregnant than those from non-pregnant cows (P<0.05). 30 ng/ml IFN-τ significantly increased 11-HSD1 activity and cortisol secretion in endometrial explants, and in both stromal and epithelial cells compare to control (P<0.05). The overall results suggest that the level of cortisol is locally regulated by INF-τ in the pregnant bovine endometrium by up-regulating 11-HSD1 expression and activity. Based on our previous and present findings, IFN-τ seems to suppress PGFα secretion by up-regulating cortisol activation, resulting in the maintenance of the CL during early pregnancy in cattle. Supported by Grants-in-Aid for Scientific Research from the Polish Ministry of Sciences and Higher Education (N308 3279 33) and Japan Society for the Promotion of Science (JSPS, No.18380166). MM, DJS and KO were supported by the Japanese-Polish Joint Research Project under the agreement between JSPS and Polish Academy of Sciences. (poster)

EG-VEGF Protein Expression in Superovulated Buffalo Corpus Luteum.

Endocrine gland derived endothelial growth factor (EG-VEGF) also termed prokinectin 1(PK-1), is a recently identified protein, which expression has been described in a variety of tissues, including steroidogenic glands, gastrointestinal tract, and nervous system. EG-VEGF is a multifunctional factor: in bovine corpus luteum (CL) it induces endothelial cell proliferation and permeability. Superovulation has been shown to promote important morphologic and functional changes in buffaloes CL: gonadotropins stimulates progesterone synthesis and induces the expression of angiogenic factors as VEGF and bFGF increasing vascular permeability. Based on these data, we hypothesized that EG-VEGF is present in buffalo CL and that superovulatory treatment influences its expression. To test these hypotheses, we used 12 water buffaloes corpora lutea collected on day 6 post ovulation: six from non-treated (control group) and six from animals submitted to superovulatory treatment. Superovulation was carried out using repeated dosis of follicle-stimulating hormone (FSH). EG-VEGF protein expression was assessed by immunohistochemistry and western blotting (WB). Data were analyzed by Student's t test and differences were considered significant at P < 0.05. A positive immunostaining was detected in the cytoplasm of luteal and endothelial cells, but stroma cells as well as pericytes were not stained for EG-VEGF. Western blots of buffalo CL protein extracts were able to detect one band, at approximately 12 kDa, which correspond to EG-VEGF. In both immunohistochemistry and densitometry, we observed a higher expression of EG-VEGF in CL of superovulated buffaloes in relation to control group (P<0.05). Our data demonstrated that EG-VEGF protein is expressed in buffaloes CL, and that FSH stimulates its expression. Our data agree with previously studies, in which one has demonstrated that hCG stimulates the synthesis of EG-VEGF in human luteinized granulosa cells. In conclusion, the presence of EG-VEGF in CL, mainly in luteal and endothelial cells, points towards a paracrine or even autocrine role of this growth factor in modulating CL function. Moreover, the increased expression observed in superovulated CL may be related to increased vascularization and hormone production, features already described for this model. (poster)

Prolactin independent rescue of mouse corpus luteum life span: identification of prolactin and luteinizing hormone target genes

The corpus luteum (CL) plays a central role in the maintenance of pregnancy in rodents, mainly by secreting progesterone. Female mice lacking prolactin (PRL) receptor (R) are sterile due to a failure of embryo implantation, which is a consequence of decreased luteinizing hormone (LH) receptor expression in the CL and inadequate levels of progesterone. We attempted to treat PRLR(-/-) females with human chorionic gonadotropin (hCG) and showed a de novo expression of LHR mRNA in the corpora lutea. Binding analysis confirmed that the LHR in hCG-treated PRLR(-/-) animals was functional. This was accompanied with increased expression of steroidogenic enzymes involved in progesterone synthesis. Despite these effects, no embryo implantation was observed because of high expression of 20alpha-hydroxysteroid dehydrogenase. To better appreciate the molecular mechanisms underlying maintenance of the CL, a series of mRNA expression-profiling experiments was performed on isolated corpora lutea of PRLR(-/-) and hCG-treated PRLR(-/-) mice. This approach revealed several novel candidate genes with potentially pivotal roles in ovarian function, among them, p27, VE-cadherin, Pten, and sFRP-4, a member of the Wnt/frizzled family. This study showed the differential role of PRL and LH in CL function and identified new targets of these hormones in luteal cells.

Effects of active immunization against growth differentiation factor 9 and/or bone morphogenetic protein 15 on ovarian function in cattle

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15 in vivo on ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to <25% of that in the controls. In conclusion, immunization against GDF9 and BMP15, alone or together, altered follicular development and ovulation rate in cattle. Thus, as has been observed in sheep, both GDF9 and BMP15 appear to be key regulators of normal follicular development and ovulation rate in cattle.

Differential luteolytic function between the physiological breeding season, autumn transition and persistent winter cyclicity in the mare

There is a well-documented increase in luteolytic failure, resulting in spontaneously prolonged corpus luteum (SPCL) function, during estrous cycles of horses in autumn. The cause of this phenomenon may be due to seasonal alterations in PGF 2α and/or in prolactin (PRL) secretion around luteolysis. To investigate this, progesterone (P4), 13, 14-dihydro, 15-keto PGF 2α (PGFM) and PRL concentrations were compared between summer and autumn estrous cycles during natural luteolysis and luteolysis induced by benign uterine stimulation. A single estrous cycle from mares in June–July ( n = 12) was compared to multiple estrous cycles from these 12 mares plus 8 additional mares in September through December. Reproductive behavior was monitored by bringing a stallion in close proximity to the mare and ovarian events by ultrasonography. Blood was collected via jugular cannula every 6 h from d 13 to 17 post-ovulation in untreated control mares ( n = 8 summer, n = 9 autumn). In treated mares, blood collection occurred at 0, 15, 30, 45, 60, 90, 120, 180 and 240 min followed by 6 h intervals for a total of 5 d following intrauterine saline infusion on d 7 ( n = 4 summer, n = 11 autumn). Mares failing to return to estrus for 30 d received intrauterine saline and the described intensive blood sampling protocol on d 30. Progesterone and PRL were determined on daily samples and PGFM on frequent plasma collections by RIA. Duration of ovarian luteal and follicular phases, P4 and PRL concentrations and PGFM secretion around luteolysis were compared between treatments and seasons by ANOVA. Mean P4 declined from June to December in all groups. Pulses of PGFM were detected on d 13–17 in controls and d 7–11 in saline-infused mares. Pulse patterns were not different between groups. The incidence of SPCL increased during autumn in the control group. PGFM pulses were absent on d 13–17 in mares with SPCL, but PGFM pulses could be induced in these mares by saline infusion at d 30. Autumn PGFM profiles were unchanged during spontaneous or saline-induced luteolysis compared with summer. Circulating PRL increased around natural or induced luteolysis. These results provide evidence that changes in luteal function during the autumn transition are not the result of alterations in the ability of the uterus to produce PGF 2α nor due to changed CL sensitivity to PGF 2α. We conclude that seasonal changes in luteolytic function are caused by an alteration in the signal for PGF 2α release.

Serum biomarkers of tubal ectopic pregnancy: current candidates and future possibilities

Ectopic pregnancy remains a considerable cause of maternal morbidity and mortality worldwide. Currently, it is diagnosed using a combination of transvaginal ultrasound and serial serum beta-human chorionic gonadotrophin levels. Diagnosis is often delayed and these tests are time-consuming and costly, both psychologically to the patient and financially to health services. The development of a biomarker that can differentiate a tubal ectopic from an intrauterine implantation is therefore important. In the pre-genomic era, a one-by-one scientific approach has revealed over 20 candidate biomarkers that could be used as a test to diagnose ectopic pregnancy although at present their clinical utility is very limited. These biomarkers cluster into themes: markers of abnormal embryo/trophoblast growth, markers of abnormal corpus luteum function, markers of a growing pregnancy in the Fallopian tube, markers of inflammation and peritoneal irritation, and uterine markers of normal implantation. It is likely that this thematic approach will facilitate the identification of newer biomarkers using microarray technology and inform the development of investigative paradigms using multiple markers at the time of presentation.

Down-regulation of genes encoding steroidogenic enzymes and hormone receptors in late preovulatory follicles of the cow coincides with an accumulation of intrafollicular steroids

The transformation of the dominant follicle into a functional corpus luteum is accompanied by a profound molecular and morphological reorganization of somatic cell layers. Several studies have focused on gene expression during early processes of follicular differentiation as it relates to recruitment and selection of dominant follicles. However, little information exists on changes of gene expression profiles in late preovulatory follicles. This lack of information is addressed here to elucidate molecular mechanisms behind the LH-induced transition from the large dominant estrogen-active to the preovulatory follicle, an intermediate stage toward full luteinization. Transcripts encoding key molecules for the biosynthesis of steroid hormones and prostaglandins, as well as receptors for gonadotropic and growth hormones ( Star, Cyp11a1, Hsd3b, Cyp17, Cyp19, Ptgs2, Fshr, Lhr, and Ghr), were quantified by real-time polymerase chain reaction (PCR) in the granulosa and theca of large dominant and late preovulatory follicles. The steroid hormones progesterone (P4) and estradiol-17β (E2) were monitored to distinguish estrogen-active and estrogen-inactive follicles. We found that (1) independent of the follicular stage, the gene expression profile was very different in granulosa and theca; (2) the abundance of several key transcripts was lower in estrogen-inactive, compared with estrogen-active, dominant follicles; (3) in the granulosa of late preovulatory follicles, transcripts encoding steroidogenic enzymes and hormone receptors were largely down-regulated, whereas (4) progesterone and E2 were found at high concentrations in the follicular fluid. Collectively, our data show that late preovulatory follicles have a transient and unique gene expression profile and are clearly different from both the preceding and subsequent (follicular and luteal, respectively) stages.

Expression and Localization of Gap Junctional Connexins 26 and 43 in Bovine Periovulatory Follicles and in Corpus Luteum During Different Functional Stages of Oestrous Cycle and Pregnancy

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1-3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8-12 were injected with PGF2alpha analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.

Expression of VEGF-receptor system in conceptus during peri-implantation period and endometrial and luteal expression of soluble VEGFR-1 in the pig

In view of the importance of vascular events observed during gestation, it was hypothesized that the VEGF-receptor system plays a critical role during early pregnancy and maternal recognition of pregnancy in pigs. This hypothesis was tested by examining the expression of the VEGF-receptor system in the porcine conceptus. Additionally, the endometrium, corpus luteum (CL) and embryos were studied for the expression of soluble VEGF receptor 1 (sVEGFR-1), the strong endogenous antagonist of VEGF. The expression patterns show that VEGF164 mRNA levels increase gradually in line with conceptus development, whereas VEGF120 and VEGFR-2 remain unchanged during the peri-implantation period. Interestingly, elevated VEGFR-1 expression was observed in conceptuses on days 15–16 of gestation ( P < 0.05). Comparison of the endometrial sVEGFR-1 mRNA expression revealed up-regulation on days 12 and 15–16 of pregnancy ( P < 0.01 and P < 0.05, respectively). Furthermore, increased sVEGFR-1 levels were observed on day 12 of the estrous cycle in the CL ( P < 0.05). Concluding, it seems that conceptus-derived VEGF164 plays crucial role in peri-implantation vascular events in pigs. These results support a potential role of VEGFR-1 in the proper growth and development of porcine conceptus during pregnancy. Moreover, expression patterns of sVEGFR-1 in the endometrium of pregnant pigs suggest that it may participate in vascular remodeling important for successful implantation. Finally, luteal sVEGFR-1 may be involved in the maintenance of CL function whenever pregnancy occurs in pigs.

Luteal-phase endocrinology

The corpus luteum is formed from the pre-ovulatory follicle under the action of the mid-cycle LH surge. LH is the main luteotrophic hormone in women controlling luteal structure and function during the normal menstrual cycle. Local factors, however, including progesterone are also involved. If conception does not take place, luteolysis occurs as a physiological apoptotic process. Human chorionic gonadotrophin, secreted after implantation, is able to rescue the corpus luteum and extend its lifespan. In ovulation-induction cycles, the negative feedback effect of the ovarian steroids on the pituitary is markedly potentiated, leading to the suppression of endogenous LH secretion during the whole menstrual cycle. The marked suppression of LH secretion disrupts corpus luteum function regardless of the treatment regimen. Summary for lay readers The corpus luteum is formed from the ovulated follicle under the influence of the endogenous LH surge. LH is the principal hormone that stimulates the production of progesterone and supports corpus luteum function in women. If conception fails, regression of corpus luteum takes place, a process named luteolysis. The causes of luteolysis in humans are not known. Nevertheless, the function of the corpus luteum is rescued when conception occurs by human chorionic gonadotrophin. In IVF programmes, ovarian stimulation leads to a marked suppression of LH secretion that disrupts corpus luteum function.

Effect of β-carotene on Ovarium Functions and Ovsynch Success in Repeat Breeder Cows

Summary This study was carried out to determine the effect of β-carotene on ovarian function and pregnancy rates in repeat breeder cows. Twenty-six repeat breeder Holstein cows were randomly assigned into control (n=11) and carotene (n=15) groups. Cows were fed with diet in the form of total mixed ration (39.80% neutral detergent fiber and 16.20% crude protein of dry matter). Treatment cows received an intramuscular injection of β-carotene (1 mg/kg) simultaneously with the first GnRH injection. Largest follicle (P<0.05) and corpus luteum diameters (P<0.01) together with serum progesterone (P<0.01), β-carotene (P<0.05) and vitamin A (P<0.01) levels were all significantly higher in the carotene group on the day of PGF2α injection. At the second GnRH injection, largest follicle diameter (P<0.01), serum estradiol (P<0.01), β-carotene (P<0.01), and vitamin A (P<0.01) levels were again higher in the carotene group. Serum estradiol (P<0.01) and β-carotene (P<0.05) levels were still significantly higher in the carotene group at the time of AI. Ovulation response did not differ between groups after both first and second GnRH injection. The pregnancy rate was 27.2% in the control group, and 33.3% in the carotene group but this difference was not statistically significant. In conclusion, it was suggested that β-carotene did not have any significant effects on ovulation or pregnancy rates in ovsynch-treated repeat breeder cows; however it increased serum estradiol and progesterone levels due to increased follicle size and corpus luteum functionality, respectively.

146. MACROPHAGES ARE ESSENTIAL FOR MAINTENANCE OF EARLY PREGNANCY THROUGH REGULATION OF CORPUS LUTEUM FUNCTION

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis [1, 2]. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion [3]. To determine the impact of macrophage ablation during early pregnancy, we utilised the macrophage specific CD11b-DTR diphtheria toxin receptor (DTR) transgenic mouse to cause transient systemic ablation of macrophages via the administration of DT (25 ng/g). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated and implantation sites were counted. Ablation of macrophages on day 1 pc (n=13 wild-type; n=9 CD11b-DTR) or day 4 pc (n=6 CD11b-DTR; n=9 wild-type) caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while wild-type mice maintained viable pregnancies [mean ± SEM implantation sites = 5.0 ± 1.4 (day 1 treated), and 6.0 ± 1.9 (day 4 treated)]. Serum progesterone was analysed 24 h following macrophage ablation. A single DT injection on day 3 pc significantly reduced serum progesterone (P4) levels [n=7 wild-type P4 (ng/ml)= 29.6 ± 3.3; n=8 CD11b-DTR = 11.1 ± 2.1]. The administration of exogenous P4 (2 mg) on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice (n=6; implantation sites = 7.8 ± 1.6), while no pregnancies remained viable in DT-treated mice administered vehicle only (n=9). In conclusion, luteal insufficiency appears to be the cause of pregnancy failure following macrophage ablation. These data indicate a critical role for macrophages in corpus luteum function in early pregnancy.

Luteolysis and pregnancy outcomes in dairy cows after treatment with estrumate or lutalyse (2009)

In Experiment 1, lactating dairy cows (n = 1,230) in 6 herds were treated with 2 injections of prostaglandin F2α(PGF2α) 14 days apart (Presynch), with the second injection administered 12 to 14 days before the onset of a timed AI protocol (Ovsynch). Cows were inseminated when detected in estrus after the Presynch PGF2αinjections. Cows not inseminated were enrolled in the Ovsynch protocol and were assigned randomly to be treated with either Estrumate or Lutalyse as part of a timed artificial insemination (AI) protocol. Blood samples were collected before treatment injection (0 hour) and 48 and 72 hours later. In cows having progesterone concentrations ≥1 ng/mL at 0 hour and potentially having a functional corpus luteum (CL) responsive to a luteolytic agent, Lutalyse increased (P < 0.05) luteal regression from 83.9 to 89.3%. Despite a significant increase in luteolysis, pregnancy rate per AI did not differ between treatments. Fertility was improved in both treatments in cows having reduced progesterone concentrations at 72 hours and in those showing signs of estrus. In Experiment 2, an ovulation resynchronization (Ovsynch-Resynch) program was initiated with gonadotropin-releasing hormone (GnRH) or saline in 427 previously inseminated lactating dairy cows of unknown pregnancy status in 1 herd. Seven days later, pregnancy was diagnosed and nonpregnant cows were blocked by number of CL and assigned randomly to receive Estrumate or Lutalyse. Diameter of each CL was recorded and blood samples were collected at 0 and 72 hours after treatment to assess serum progesterone. A fixed-time AI was given at 72 hours after treatment and approximately 16 hours after a GnRH injection to induce ovulation. Lutalyse increased(P < 0.05) luteal regression from 69.1 to 78.5% regardless of the number of CL present or the total luteal volume per cow exposed to treatment. Pregnancy rate per AI did not differ between treatments. Although Lutalyse was slightly more effective than Estrumate in inducing luteolysis in lactating dairy cows exposed to an Ovsynch or Ovsynch-Resynch protocol, resulting pregnancy outcomes did not differ between products.; Dairy Day, 2009, Kansas State University, Manhattan, KS, 2009; Dairy Research, 2009 is known as Dairy Day, 2009

Changes in the Vasculature of Bovine Corpus Luteum During the Estrous Cycle and Prostaglandin F2&amp;alpha;-induced Luteolysis

To investigate the possible role of the vasculature in the local regulation of corpus luteum (CL) function, we determined the densities of capillaries and large blood vessels in the center of the bovine CL during the estrous cycle and following prostaglandin (PG) F2alpha-induced luteolysis. The CLs at the early (Days 2-3 post-ovulation), developing (Days 5-7), mid (Days 8-12), late (Days 15-17) and regressed (Days 19-21) stages were collected. In addition, the CLs were collected by transvaginal ovariectomy from 12 cows (Day 10 after ovulation), i.e., non-treated (n=3, 0 h, control), at 0.5 (n=3), 2 (n=3) and 12 h (n=3) after injection of a luteolytic dose of PGF2alpha. Immunohistochemical staining with von Willebrand Factor (specific for endothelial cells that are found in both types of blood vessels) revealed that the density of the luteal blood vessels was significantly higher at the developing and late luteal stages (P<0.05) than at the other stages, whereas the number of larger blood vessels (those stained with alpha-smooth muscle actin) was higher at the late and regressed luteal stages (P<0.05) than at the other stages. Furthermore, both the density of blood vessels and the number of blood vessels with smooth muscle were significantly higher in the CLs obtained at 2 h and 12 h after PGF2alpha administration (P<0.05) than in those without PGF2alpha treatment. These results suggest that the number of blood vessels with smooth muscle per unit area in the regressing CL increased as a result of losing steroidogenic cells and capillaries. The overall results demonstrate that the capillaries disappeared earlier than the large blood vessels during structural luteolysis and suggest that the loss of capillaries in the CL results in a reduced supply of nutrients and oxygen to luteal cells followed by cell death.

THE EXPRESSION OF APELIN AND ITS RECEPTOR APJ DURING DIFFERENT PHYSIOLOGICAL STAGES IN THE BOVINE OVARY

Recent studies implicate that apelin and its receptor APJ may have important role for the modulation of angiogenesis. The aim of this study was to further characterise the regulation of apelin/APJ system in bovine ovary. Experiment 1: corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of gravidity (month <3, 3-5, 6-7 and >8). Experiment 2: Follicles during maturation were divided into granulosa cells (GC) and theca interna (TI) and were examined separately. Classification of follicles occurred by follicle size and oestradiol-17beta (E2) concentration in the follicular fluid (FF) (<0.5 ng/ml, 0.5-5 ng/ml; 5-40 ng/ml; 40-180 ng/ml; >180 ng/ml). Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, the expression level of apelin during the oestrous cycle was significantly higher compared to the one during pregnancy. Apelin mRNA levels were always high during the cycle with a tendency of decrease after CL regression. The APJ mRNA in the CL was significantly up regulated on days 5-7 and 8-12 followed by a decrease on days 13-16, and further on days >18. The expression of APJ does not show any significant regulation in the CL throughout pregnancy. The expression of apelin and APJ was not statistically regulated in GC, but was significantly up regulated in follicles with an E2 concentration of more than 5 ng/ml and showed an increase according to growth and maturation of follicles. In conclusion, our data suggest that apelin/APJ system is involved in the mechanism regulating angiogenesis during follicle maturation as well as during CL formation and function in the bovine ovary.

Production of Trophoblastic Vesicles Derived From Day 7 and 8 Blastocysts of In Vitro Origin and the Effect of Intrauterine Transfer on the Interestrous Intervals in Japanese Black Heifers

This study was undertaken to produce trophoblastic vesicles (TVs) by using blastocysts of in vitro origin and to estimate the effect on the interestrous interval after transfer of 4 TVs into the uteri of heifers on Day 7. Morphological examination under a stereoscopic microscope revealed that the total formation rate of TVs prepared from IVP expanded blastocysts was 80.5% and that there was no difference in the formation rates of TVs derived from blastocysts between Day 7 (83.5%) and Day 8 (78.5%). After intrauterine transfer of TVs, observation of the corpus luteum (CL) by transrectal ultrasonography together with measurement of the plasma progesterone concentration confirmed that 2 of 4 recipients (50%) had a longer interestrous interval, 33.5 and 35.0 days, while the other 2 recipients had normal cycles, 20.0 and 24.5 days. In the control group transferred D-PBS, all 4 heifers had a normal cycles, 24.0-24.5 days. Consequently, the average number of days after intrauterine transfer of TVs compared with the 2 consecutive cycles just before the treatment was longer than in the controls (6.1 +/- 2.4 days vs. -0.8 +/- 1.1 days, P<0.05). These results indicate that preparation of TVs from blastocysts of in vitro origin is a useable method and that TVs from blastocysts may have the capacity to maintain CL function after intrauterine transfer.

&lt;i&gt;In Vitro&lt;/i&gt; Assessment of Progesterone and Prostaglandin E&lt;sub&gt;2&lt;/sub&gt; Production by the Corpus Luteum in Cattle Following Pharmacological Synchronization of Estrus

We studied the secretory function of the corpus luteum (CL) in cows following different estrus synchronization protocols. Estrus was synchronized using one (n=4) or two injections (n=5) of prostaglandin F(2alpha) (PGF(2alpha); dinoprost), two injections of different analogues of PGF(2alpha) (aPGF(2alpha)), luprostiol (n=5) and cloprostenol (n=5), at eleven-day intervals, a gestagen implant (norgestomet, n=5, for 10 days) or norgestomet together with a subsequent dinoprost injection on the day of implant removal (n=5). CL samples were collected by ovariectomy on Day 7-8 of the estrous cycle. Luteal strips were stimulated with LH (100 ng/ml) or prostaglandin E(2) (PGE(2), 10(-6)M) for 24 h in culture media. The progesterone (P(4)) and PGE(2) concentrations in the media were measured by enzyme immunoassay. In the control CL (spontaneous estrus; n=5), LH and PGE(2) stimulated P(4) and PGE(2) (P<0.001). The effects of both factors on P(4) were reduced in the CL following dinoprost- and cloprostenol-synchronized estrus (P<0.05) and were absent in the luprostiol-synchronized CL (P>0.05). In the norgestomet-synchronized CL, the stimulatory effects of LH and PGE(2) were higher compared with the CL synchronized by aPGF(2alpha) (P<0.05). Pharmacological manipulation of the estrous cycle using aPGF(2alpha) may cause lower P(4) secretion. Estrus synchronization inhibited CL sensitivity to luteotropic factors. Therefore, attention should be focused on the estrous synchronization method in both in vivo and in vitro studies of CL functions in cattle.

Comparison of Two Short Estrus Synchronization Protocols and Timed Artificial Insemination in Dairy Cows

Leyva, C. and Mellado, M. 2008. Comparison of two short estrus synchronization protocols and timed artificial insemination in dairy cows. J. Appl. Anim. Res., 34: 105–108. To compare two short protocols for synchronization of estrus and timed-artificial insemination (TAI), 79 pluriparous non-pregnant Holstein cows with active corpus luteum (CL) were selected from a commercial dairy farm in northern Mexico and allotted to one of two treatments in a completely randomised design. Group 1 cows (n=39) received an injection of 15 mg PGF2α and 24 h later received 1 mg estradiol cypionate (ECP). Two days later cows were inseminated by appointment. Cows in group 2 received an injection of 100 fig GnRH 48 h after the PGF2α injection and underwent a timed AI at 16–20 h after the GnRH injection. At the moment of CL diagnosis, blood samples were collected from all cows and analyzed for progesterone, to corroborate CL diagnosis. Pregnancy rates of cows in estrus were similar for both treatment groups with similar percentage of cows displaying intense estrus. The present study showed that estrus of lactating dairy cows can be successfully synchronized with short synchronization protocols using either GnRH or ECP, although the inaccuracy of rectal palpation for determining the presence of a functional corpus luteum lead to lower pregnancy rates than those attained with traditional protocol currently available to dairy producers.