Luteal Lifespan Research Articles

Corpora lutea of pregnant and pseudopregnant domestic cats reveal similar steroidogenic capacities during the luteal life span

In domestic cats, luteal phases of pregnancy and pseudopregnancy (non-pregnant luteal phase) differ in the course and level of plasma progesterone (P4). Therefore, we assumed differences in luteal steroidogenic capacities. Here we present a comprehensive analysis of intraluteal steroid biogenesis in the domestic cat. We quantitatively measured relative mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYP), hydroxysteroid dehydrogenases (HSD), steroid reductase (SRD) and enzymes involved in sulfoconjugation of steroids, i.e. sulfotransferase (SULT) and sulfatase (STS). Protein expression was analysed by Western Blot for HSD3B. Additionally, intraluteal steroid contents were determined. During the pseudopregnant luteal phase, expression of STAR (p=0.005), HSD3B1 (p<0.0001), CYP19A1 (p<0.0001) and HSD17B7 (p=0.008) decreased from formation of the corpus luteum (CL) onwards. HSD3B protein expression was highest in the development/maintenance stage of CL and declined during the subsequent luteal phase of pregnancy and pseudopregnancy. This was in accordance with decreasing intraluteal levels of P4, oestrogens and androgens. In contrast, expression of SRD5A1 (p<0.001) increased with progression through stages of the pseudopregnant CL, being indicative of P4 metabolism via an alternate pathway to dihydrotestosterone (DHT). Compared to the formation stage, expression of SULT1E1 was higher in all other luteal stages of pseudopregnancy (p=0.004), implying a potential sulfoconjugation of oestrogens. Expression of CYP11A1 and CYP17A1 was unaffected by the luteal stage (p>0.05), suggesting a permanent capacity of cat CL to convert progestogens via androgen and oestrogen pathways. In general, mRNA expression profiles of steroidogenic enzymes during the pregnant luteal phase reflected the pseudopregnancy profiles. Intraluteal oestrogen (p<0.0001) and androgen (p=0.008) levels were higher in the formation stage compared to the following luteal stages of pseudopregnancy. Concentrations of P4 were higher in the development/maintenance compared to the regression stages (p=0.01). We conclude that cat CL of the same histomorphological stage are characterised by identical steroidogenic capacities independently of an on-going pregnancy.

Life threatening corpus luteal hemorrhage

Introduction Ovulation is a physiological monthly event in women of reproductive age. Corpus luteum hemorrhage may occur spontaneously or often triggered by coitus, trauma, exercise, or vaginal examination [1]. The risk of hemorrhagic complications of ovulation starts on the ovulation day and extends throughout corpus luteal life span, which is 14

118 DEEP SEQUENCING OF THE PORCINE ENDOMETRIAL TRANSCRIPTOME ON DAYS 12 AND 14 OF PREGNANCY

Establishment of pregnancy in mammals requires prolongation of luteal life span for sustained progesterone (P4) production. P4 stimulates secretory functions of the endometrium required for conceptus growth and development and is essential for endometrial receptivity. The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Day 11 and Day 12 of gestation. With trophoblast elongation production of estrogen, the porcine embryonic pregnancy recognition signal, by the conceptus increases. A complex interplay of estrogen signaling and prostaglandin (PG) metabolism in the endometrium finally results in prevention of luteolysis. Conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy, preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of maternal recognition of pregnancy and initial placentation, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding non-pregnant controls (n = 4) was performed on Day 12 and Day 14 after ovulation using Illumina RNA-Seq (Illumina Inc., San Diego, CA, USA). Between 21 and 36 million 76-bp single-end reads were produced per sample. Reads were mapped with TopHat to the porcine genome assembly Sscrofa10.2 and relative expression values were calculated based on the National Center for Biotechnology Information’s gene annotation for the analysis of differential gene expression. Statistical analysis revealed more than 3500 differentially expressed genes (DEG) for Day 12 and almost 2500 DEG for Day 14 of pregnancy (DESeq, FDR 1%). Differential expression of selected genes was validated by the use of quantitative real-time PCR (RT-PCR). Comparison of the RNA-Seq data from Day 12 and Day 14 of pregnancy based on a comparison of the DEGs and of overrepresented functional categories revealed distinct differences in gene regulation, reflecting the different functions of the endometrium during these stages; that is, recognition of pregnancy on Day 12 and preparation for conceptus implantation on Day 14. The data from Day 14 was also compared to the published microarray data set from Day 14 of pregnancy (Østrup et al. 2010 Biol. Reprod.) and other sets of DEG by the use of gene set enrichment analysis (GSEA). Overall, a very good agreement with the results of the previous microarray study was found. Furthermore, a significant overlap with genes upregulated in bovine endometrium on Day 18 of pregnancy was found for the genes upregulated in porcine endometrium on Day 14. More than half of these genes were known interferon-regulated genes. In conclusion, RNA-Seq revealed distinct transcriptome changes on Day 12 and Day 14 of pregnancy in porcine endometrium associated with MRP and initiation of implantation.

122 OXYTOCIN-INDUCED PROLONGED DIOESTRUS DOES NOT DOWNREGULATE THE EXPRESSION OF PROSTAGLANDIN-ENDOPEROXIDASE SYNTHASE 2 OR OXYTOCIN RECEPTOR IN EQUINE ENDOMETRIUM

Lifespan of the corpus luteum is regulated through the release of PGF2α from the endometrium. The endocrine factors regulating the timed release of prostaglandin during luteolysis and the abrogation thereof during pregnancy is only partially understood in the mare. Vanderwall et al. (2007 J. Am. Vet. Med. Assoc. 231, 1864–1867) reported that twice-daily administration of oxytocin during early dioestrus results in prolongation of luteal function. We hypothesised that the aforementioned repeated administration of oxytocin during early dioestrus results in downregulation of oxytocin receptor transcript while prostaglandin-endoperoxide synthase 2 transcript abundance is unaffected. The current study, therefore, compared the expression oxytocin receptor (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) in endometrial samples obtained 16 days after ovulation from nonpregnant mares (n = 5), pregnant mares (n = 5), and mares displaying prolonged dioestrus after the repeated administration of oxytocin (n = 6). Prolonged dioestrus was observed in all mares treated with oxytocin, evident by plasma progesterone levels above 6 ng mL–1 at the time of sample collection. Using quantitative real-time PCR analysis, PTGS2 showed a 4.37-fold lower abundance in pregnant v. nonpregnant mares (P = 0.009), while OXTR abundance was not affected by pregnancy status. Repeated administration of oxytocin did not lower expression levels of PTGS2 or OXTR when compared to levels observed in nonpregnant mares. As hypothesised, PTGS2 abundance was not affected by the treatment with oxytocin. Furthermore, we failed to prove the hypothesis that repeated administration of oxytocin leads to lowered expression of OXTR 16 days after ovulation in mares displaying prolonged dioestrus following repeated administration of oxytocin. A regulation of the oxytocin receptor at the post-transcriptional level, such as uncoupling of the receptor from its G-protein or possibly phosphorylation of the receptor, cannot be excluded and might account for the observed prolongation of luteal lifespan.

Endocrine and Molecular Control of Luteal and Placental Function in Dogs: A Review

In the domestic dog (Canis familiaris), the corpus luteum (CL) is the only source of progesterone (P4) in non-pregnant and pregnant animals. The progesterone secretion profiles are almost identical in both conditions until the last third of the luteal phase when the gradual P4 decline turns into a steep drop in pregnant bitches, indicating the onset of parturition. Consequently, the length of the CL-phase in non-pregnant dogs exceeds the luteal lifespan in pregnant animals. The canine CL-function is regulated by many species-specific regulatory mechanisms, the most intriguing of which is the reported independence of gonadotropic support during the first third of dioestrus. Recently, PGE2 has been proposed as one of the most important luteotropic factors acting locally during this time, but afterwards prolactin (PRL) appears to be the main luteotropic factor. Luteal regression/luteolysis occurs, however, in spite of an increased gonadotropic support. Lately, by demonstrating the expression of PRL-receptor (PRLr), a new insight into possible regulatory mechanisms has indicated that the supply of P4 could be controlled upstream of the steroidogenic machinery at the level of PRLr expression and/or function, subsequently leading to the functional suppression of the steroidogenic machinery. An endogenous source of a luteolytic agent is apparently lacking, implicating the luteal regression in non-pregnant bitches as a passive, degenerative process even if the PGF2α-receptor is constitutively expressed in canine CL. This is in contrast to pregnant dogs in which prepartum luteolysis seems to be an active process of CL destruction by PGF2α of utero/placental origin targeting the luteal PGF2α-receptor.

Quantification of blood volume and vascular perfusion in the corpus luteum of rhesus macaques during the menstrual cycle

OBJECTIVE: To determine changes to luteal vascular flow and volume in rhesus monkeys using dynamic contrast enhanced ultrasound (d-CEU). DESIGN: In vivo, repeated measures. MATERIALS AND METHODS: Hormonal status of 8 adult cycling female macaques was monitored to detect the day of ovulation. D-CEU with Definity (Lantheus) microbubble perfusion was performed on the corpus luteum (CL) at the early, mid, mid-late and late luteal phase (LP; Mol Reprod Dev, 76: 367-378, 2009), as recently described for the uterus (Fertil Steril 95:1316, 2011). Vascular parameters were quantified in d-CEU video captures using a region of interest (ROI) focused on the CL. Video intensity (VI) was normalized to brachial artery flow to provide an estimate of blood volume (BV); change in VI (DVI/sec) was used to determine vascular perfusion rate (VP). RESULTS: Significant changes in BVand VP (P<001 and P1⁄40.07) were observed between stages of the LP. BV was negatively correlated with luteal day (P<0.02). In contrast, serum progesterone (P) levels correlated positively with BV (P1⁄40.04) and VP (P1⁄40.001). P area under curve (AUC) also positively correlated with AUC for BV (P1⁄40.03). In typical LPs, BV was highest during the early LP, before decreasing 3-fold in late LP CL. In contrast, VP peaked at mid LP, with values 5 and 11-fold greater than early and late LP, respectively. Two females were identified with luteal insufficiency based on peak P levels during the mid and mid-late LP (2.3 0.2 ng/ml), whereas 5 had normal LPs (6.2 1.2 ng/ml). Another female was identified with a luteal cyst on the contralateral ovary, and a functional CL that regressed before P levels declined. Insufficient LPs trended toward lower peak BVand VP, compared to typical LPs. CONCLUSION: D-CEU provides a non-invasive measurement of the dynamics of CL blood volume and flow during the luteal lifespan in the menstrual cycle. Reduced vascular perfusion is evident in animals with reduced P secretion and in animals with aberrant luteal function. Supported by: P51OD011092, R01HD020869 (RLS).

Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule

Is decorin (DCN), a putative modulator of growth factor (GF) signaling, expressed in the primate ovary and does it play a role in ovarian biology? DCN expression in the theca, the corpus luteum (CL), its presence in the follicular fluid (FF) and its actions revealed in human IVF-derived granulosa cells (GCs), suggest that it plays multiple roles in the ovary including folliculogenesis, ovulation and survival of the CL. DCN is a secreted proteoglycan, which has a structural role in the extracellular matrix (ECM) and also interferes with the signaling of multiple GF/GF receptors (GFRs). However, DCN expression and action in the primate ovary has yet to be determined. Archival human and monkey ovarian samples were analyzed. Studies were conducted using FF and GC samples collected from IVF patients. Immunohistochemistry, western blotting, RT-PCR, quantitative RT-PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) studies were complemented by cellular studies, including the measurements of intracellular Ca²⁺, reactive oxygen species (ROS), epidermal GF receptor (EGFR) phosphorylation by DCN and caspase activity. Immunohistochemistry revealed strong DCN staining in the connective tissue and follicular thecal compartments, but not in GCs of pre-antral and antral follicles. Pre-ovulatory follicles could not be studied, but DCN was associated with connective tissue of CL samples and the cytoplasm of luteal cells. DCN expression in monkey CL doubled (P < 0.05) towards the end of the luteal lifespan. DCN was found in human FF obtained from IVF patients (mean: 12.9 ng/ml; n = 20) as determined by ELISA. DCN mRNA and/or protein were detected in freshly isolated and cultured, luteinized human GCs. In the latter, exogenous human recombinant DCN increased intracellular Ca²⁺ levels and induced the production of ROS in a concentration-dependent manner. DCN, like epidermal GF, phosphorylated EGFR significantly (P < 0.05) and reduced the activity of caspase 3/7 in cultured GCs. The data indicate the expression of DCN in the theca of growing follicles, in FF of ovulatory follicles and in the CL. Therefore, DCN may exert paracrine actions via GF/GFR systems in multiple ovarian compartments. Functional studies were performed in cultures of human luteinized GCs, which are an apt model but may not fully mirror the pre-ovulatory GC compartment or the CL. Other human ovarian cells, including the thecal cells, were not available. In accordance with its evolving roles in other organs, ovarian DCN is an ECM-associated component, which acts as a multifunctional regulator of GF signaling in the primate ovary. DCN may thus be involved in folliculogenesis, ovulation and the regulation of the CL survival in primates. This study was supported by Deutsche Forschungsgemeinschaft (DFG) MA1080/17-3 and in part DFG MA1080/21-1 (to AM), NIH grants HD24870 (S.R.O. and R.L.S.), the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement HD18185 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (S.R.O.) and 8P51OD011092-53 for the operation of the Oregon National Primate Research Center (G.A.D., J.D.H., S.R.O. and R.L.S).

Quantification of Immune Cells in the Primate Corpus Luteum During the Menstrual Cycle; Increases During the Late Luteal Phase Indicate a Potential Role in Luteolysis.

The potential role of the immune system in regulation of the mammalian ovary has primarily been investigated in domestic animal and rodent models; however the presence of immune cells in the human ovary has also been noted by immunohistochemistry. Recently, microarray-identified luteal mRNAs for immune regulatory components such as chemokines and cytokines, immune cell-associated genes, as well as gene signaling pathways associated with immune function were found to fluctuate during the lifespan of rhesus macaque corpora lutea (CL). To characterize immune cell populations in primate CL throughout the menstrual cycle, luteal tissue was collected from rhesus females at discrete intervals of the luteal phase, including early days 3-5 (n=3), mid days 6-8 (n=4), mid-late day 10 (n=4) and day 12 (n=3), as well as after onset of luteal regression (2 days n=2, and 3-4 days n=4, following a drop in serum progesterone levels to < 0.3 ng/ml). Luteal cells were enzymatically dispersed, counted and viability assessed. Wet weight (mg) of CL were significantly different across the luteal phase (P<0.03), but total numbers of viable cells did not differ between groups (P=0.8). Dispersed cells were then incubated with fluorescently-labeled antibodies specific for immune-cell surface proteins: CD11b (ITGAM; neutrophils including activated lymphocytes, granulocytes and monocytes), CD14 (monocytes/macrophages), CD16 (FCGR3; primarily natural killer cells as well as neutrophils and macrophages), and CD3 (CD3-epsilon/T cell receptor complex; T lymphocytes). Number of labeled cells for each CL (positive events in 10,000 cells counted) was assessed using a BD LSR II flow cytometry instrument for each antibody (FCS Express version 3, De Novo Software). A non-immune, isotype antibody matched for each cell surface specific marker was used to gate the positive cells within the CL. The number of positive events recorded for immune cells at each stage of the luteal phase was then analyzed by one-way ANOVA (SAS version 2.0). Both CD11b- and CD14-positive cells (neutrophils and macrophages) were constant until increasing significantly 3-4 days after onset of luteal regression (P<0.0001). Numbers of CD16-positive (natural killer cells) increased initially between early and mid luteal phase to plateau through the mid-late stages, until a further 3-fold increase by 3-4 days after onset of regression (P<0.005). Additionally, CD16-positive cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases, and increased to levels comparable with CD11b-positive cells during luteal regression. Presence of CD3-positive lymphocytes tended to increase 3-4 days after regression (P=0.07), but were less abundant than other cell types. These data demonstrate a remarkable increase in cells of the innate immune system, (i.e. macrophages and neutrophils) in the primate CL associated with the latter stages of luteal regression. Natural killer cells may have a dual role in the CL to promote luteal structure and function during the mid-luteal phase, whereas macrophages, neutrophils and natural killer cells may participate in structural luetolysis at the end of the luteal lifespan. Supported by NIH grant R01HD020869 (RLS) and NCRR grant P51RR000163 (Support For National Primate Research Center).

Life or Death Decisions in the Corpus Luteum

The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F(2α) initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF(2α) to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF(2α) and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.

Ovulatory response of different GnRH analogues and subsequent corpus luteum lifespan in the presence of norgestomet in Holstein heifers

Summary This study investigated the effect of GnRH analogues on inducing ovulation of dominant follicle (DF) and subsequent corpus luteum (CL) lifespan in the presence of norgestomet implant in Holstein heifers. On day 6 to 8 of the estrous cycle (day 0 of the experiment), all heifers (n=15) received norgestomet implant followed by prostaglandin injection on days 0 and 1. On day 4, group 1 (n=4) and 2 (n=4) heifers received 12.5 and 25 μg Alarelin, respectively. Heifers in group 3 (n=3) received 10 μg Buserelin. Heifers in control group (n=4) did not receive any further treatment. Norgestomet was removed on day 8 in the treatment group and kept throughout the experiment in control group. From day 0, blood sampling and ultrasonography were performed. Heifers were monitored after inducing ovulation for 2 consecutive estrous cycles. DF ovulated within 33 h after GnRH injection. Progesterone started to rise on day 3 and declined on day 7.45 ± 0.28 after GnRH injection. Heifers in treatment groups displayed estrus 7.73 ± 0.24 days after GnRH injection followed by spontaneous ovulation. The lifespan of CLs were 5.36 ± 0.28 and 16.9 ± 0.37 days after inducing ovulation of DF in the presence of norgestomet and following spontaneous ovulation, respectively (P<0.05). In conclusion, the half dose of Alarelin induced ovulation of DF and the presence of single norgestomet implant after ovulation may be associated with short luteal lifespan.

Luteal Function and Follicular Growth Following Follicular Aspiration During the Peri‐Luteolysis Period in Bos indicus and Crossbred Cattle

Follicular estradiol triggers luteolysis in cattle. Therefore, the control of follicle growth and steroidogenesis is expected to modulate luteal function and might be used as an anti-luteolytic strategy to improve embryo survival. Objectives were to evaluate follicular dynamics, plasma concentrations of estradiol and luteal lifespan in Bos indicus and crossbred cows subjected to sequential follicular aspirations. From D13 to D25 of a synchronized cycle (ovulation = D1), Nelore or crossbred, non-pregnant and non-lactating cows were submitted to daily ultrasound-guided aspiration of follicles >6 mm (n = 10) or to sham aspirations (n = 8). Diameter of the largest follicle on the day of luteolysis (7.4 ± 1.0 vs 9.7 ± 1.0 mm; mean ± SEM), number of days in which follicles >6 mm were present (2.3 ± 0.4 vs 4.6 ± 0.5 days) and daily mean diameter of the largest follicle between D15 and D19 (6.4 ± 0.2 vs 8.5 ± 0.3 mm) were smaller (p < 0.01) in the aspirated group compared with the control group, respectively. Aspiration tended to reduce (p < 0.10) plasma estradiol concentrations between D18 and D20 (2.95 ± 0.54 vs 4.30 ± 0.55 pg/ml). The luteal lifespan was similar (p > 0.10) between the groups (19.6 ± 0.4 days), whereas the oestrous cycle was longer (p < 0.01) in the aspirated group (31.4 ± 1.2 vs 21.2 ± 1.3 days). Hyperechogenic structures were present at the sites of aspiration and were associated with increase in concentration of progesterone between luteolysis and oestrus. It is concluded that follicular aspiration extended the oestrous cycle and decreased the average follicular diameter on the peri-luteolysis period but failed to delay luteolysis.

Effect of a single intrauterine administration of recombinant bovine interferon-τ on day 7 of the estrous cycle on the luteal phase length and blood profile in dairy cows

This study tested the effect of recombinant bovine interferon-tau (rboIFN-τ) on the length of estrous cycle, luteal lifespan and side effects of rboIFN-τ in the cow. A normal estrous cycle in six non-lactating cycling Holstein cows was observed (non-treated cycle), and either 2.0mg of liposomalized rboIFN-τ (treated cycle) or bovine serum albumin (BSA; placebo cycle) was infused in the uterus on day 7 of the estrous cycle (day 0=day of ovulation). Rectal temperature, heart rate and respiratory rate were recorded and blood samples were collected before and after the treatments. The length of the estrous cycle and corpus luteum lifespan in rboIFN-τ treated cycles were not significantly different from those of the non-treated and placebo cycles. In contrast, the rboIFN-τ treatment caused a transient increase in rectal temperature and a decrease in the number of peripheral lymphocytes and neutrophils after the treatment.

The Corpus Luteum of the Dog: Source and Target of Steroid Hormones?

Aim of this paper is to review our present understanding on the endocrine control of luteal function in the bitch and to add some new data generated in our laboratories in support of the hypothesis of a paracrine/autocrine role of corpus luteum (CL) derived steroid hormones. Luteal lifespan in non-pregnant dogs often exceeds that of pregnant dogs, where luteal regression terminates in a rapid luteolysis, immediately prior to parturition. In non-pregnant dogs, luteal regression occurs independently of a uterine luteolysin and in spite of increased gonadotropic support during the last third of dioestrus. The CL is the only source of progesterone (P(4)) maintaining pregnancy, and they have the capacity to synthesize oestrogens as substantiated by expression of the CYP19 (aromatase) gene observed in this study. Our data demonstrated that lutein and non-lutein cells of the canine CL express in a rather constant manner the progesterone receptor (PR) and the oestrogen receptor, classifying them as targets for an autocrine/paracrine activity of CL-derived steroids. Therefore, a functional role of P(4) within a positive loop feedback system, including StAR and 3β-hydroxysteroid dehydrogenase, has been postulated.

Intrauterine administration of plant oils inhibits luteolysis in the mare

The maternal recognition of pregnancy (MRP) signal in the mare has not been determined, although oestrogens have been proposed as a potential candidate. To determine effects of intrauterine administration of oestrogen and various oils on cyclic luteolysis in the mare. Intrauterine oestradiol or fatty acids may suppress luteolysis in the cycling mare when administered during late dioestrus. A single 1 ml dose of slow-release oestradiol (10 mg/ml) in fractionated coconut oil was infused into the uterine lumen of cycling mares on Days 6, 8, 10, 12 or 14 post ovulation (n=12 in each group). Four further groups, each of 12 mares, received an intrauterine infusion of either 1 ml of fractionated coconut oil, peanut oil, mineral oil or a slow-release preparation of oestradiol (10 mg/ml) in mineral oil on Day 10 post ovulation. Serial blood samples were assayed for progesterone concentrations to monitor luteal function. Intrauterine administration of oestradiol in fractionated coconut oil showed peak efficacy at Day 10 when luteolysis was delayed in 11/12 (92%) mares. The ability of the treatment to delay luteolysis was not significantly different when administered on Days 8 (9/12; 75%), 12 (10/12; 83%) or 14 (6/12; 50%) of dioestrus, but declined significantly when given on Day 6 (3/12; 25%). Oestradiol was not needed to initiate luteostasis since fractionated coconut oil alone or peanut oil administered at Day 10 induced the same high rate of luteal persistence (11/12; 92% for both oils). In contrast, mineral oil did not prolong luteal lifespan, either when administered alone (2/12; 17%) or combined with oestradiol (3/12; 25%). These results do not unequivocally rule out a possible involvement of embryonic oestrogens in MRP in the mare but suggest it is unlikely. The results demonstrate that plant oils can postpone luteolysis, suggesting they may modulate synthesis or release of prostaglandins from the mare's endometrium. Administration of fractionated coconut or peanut oil on Day 10 post ovulation provides an effective and practical method of prolonging luteal function ('pseudopregnancy') thereby suppressing unwanted oestrous behaviour. Further studies to elucidate the mechanism by which this is achieved may increase understanding of both luteostasis and MRP signal in the mare.

445 RELATIONSHIPS OF LUTEAL PHASE VARIABLES (PRIOR TO AI) WITH FOLLICULAR WAVES IN WATER BUFFALOES

This study was done to consider relationships of luteal phase variables (prior to AI) with follicular waves. The estrous cycles of 15 buffaloes were synchronized with 2 i.m. injections of prostaglandin F2α given 11 days apart. The buffaloes were randomly assigned to 1 of 3 treatments. Buffaloes in the control treatment received no treatment, whereas G6 buffalos received a GnRH injection between Day 5 and 7 and G16 buffalos received a GnRH injection between Day 15 and 17 of the estrous cycle (estrus = Day 0). Daily, from estrus Day 0 to the next estrus Day 23, buffaloes had their ovaries scanned by ultrasound. Blood samples were collected by tail following each ultrasound examination from estrus until next estrus (estrus = 0). Concentrations of plasma progesterone were determined by radioimmunoassay kit. The limit of detection of the assay was 0.1 45 ng mL-1 and the intra- and interassay coeffients of variation were 7.4% and 9.2%, respectively. Data were analyzed by using PROC GLM of SAS (SAS Institute, Cary, NC, USA). For comparisons between groups, the 2-sample t-test was used for continuous traits, such as size of CL or hormone concentrations. Prospective comparisons of indices of progesterone indicated that the length of luteal lifespan was longer in 3-wave than in 2-wave buffaloes (P &lt; 0.01). Plasma progesterone concentrations were similar at peak and measured as area under the curve on Day 5 through 17 preceding insemination in 2-wave (5.30 ± 0.40 ng mL-1) and 3-wave buffaloes (5.10 ± 0.20 ng mL-1). Length of the luteal phase (defined as from the day of estrus until the last day on which plasma progesterone remained &gt;2 ng mL-1) was &lt;2 days shorter in 2-wave buffaloes than in 3-wave buffaloes (15.20 ± 0.40 v. 17.10 ± 0.50 d; P &lt; 0.05). In addition, the day of peak progesterone occurred earlier in 2-wave buffaloes (13.50 ± 0.30 v. 15.30 ± 0.70 d; P &lt; 0.05).

Existence of the lymphatic system in the primate corpus luteum.

To date, there have been no detailed studies on the lymphatic system in the primate corpus luteum (CL); early reports suggested that the presence of this "secondary circulation" in luteal tissue is species-dependant. Therefore, studies were designed to determine if (a) lymphatic vessels exist, and (b) recently discovered lymphangiogenic factors and their receptor are expressed in the macaque CL during the menstrual cycle. Immunohistochemistry (IHC) detected the lymphatic endothelial cell marker, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), in some endothelial cells and vessels within the ovarian stroma and theca layer of preovulatory follicles and in the CL. Dual fluorescent IHC demonstrated that LYVE1 co-localized with another lymphatic endothelial cell marker D2-40, but a blood vascular endothelial cell marker (von Willebrand Factor, VWF) was in different cells. The numbers and staining intensity of LYVE1-positive cells in the CL appeared to increase from early to mid luteal phase, and remained elevated thereafter. RT-PCR detected cDNA fragments for mRNAs encoding VEGFC, FIGF, and their receptor FLT4 in CL. Real-time PCR analyses revealed similar patterns of VEGFC and FLT4 expression during the luteal lifespan; mRNA levels increased (p < 0.05) from early to mid luteal phase and decreased (p < 0.05) by late luteal phase. In contrast, FIGF levels were elevated initially, declined (p < 0.05) at mid luteal phase, and then increased (p < 0.05) to very late luteal phase. The data strongly suggest that lymphatic vessels are present in the primate CL, and that the VEGFC/FIGF-FLT4 system regulates lymphangiogenesis and luteal structure-function during the menstrual cycle.

517. RELATIONSHIPS OF LUTEAL PHASE VARIABLES (PRIOR TO AI) WITH FOLLICULAR WAVES IN DAIRY COWS

This study was done to consider relationships of luteal phase variables (prior to AI) with follicular waves. The estrous cycles of 10 cows were synchronized with 2 im injections of prostaglandin F2α given 11 d apart. The cows were randomly assigned to 1 of 2 treatments. Cows in the control treatment received no treatment, whereas GnRH6 cows received a GnRH injection on d 6 of the estrous cycle (estrus = d 0). Daily, from estrus d 0 to the next estrus d 23, cows had their ovaries scanned by ultrasound. Blood samples were collected by tail following each ultrasound examination from estrus until next estrus (estrus = d 0). Concentrations of plasma progesterone were determined by radioimmunoassay kit. The limit of detection of the assay was 0.125 ng/mL and the intra- and interassay coefficients of variation were 8.4% and 10.2%, respectively. Data that were analyzed by using PROC GLM of SAS. For comparisons between groups, the 2-sample t-test was used for continuous traits, such as size of cl or hormone concentrations. Prospective comparisons of indices of progesterone indicated that the length of luteal lifespan was longer in three-wave than in two-wave cows (P&lt;0.01). Plasma progesterone concentrations were similar at peak and measured as area under the curve on day 5 through 17 preceding insemination in two-wave (6.70±0.30 ng/ml) and three-wave cows (7.30±0.50 ng/ml). Length of the luteal phase (defined as from the day of estrus until the last day on which plasma progesterone remained greater than 2 ng/ml) was &lt;2 days shorter in two-wave cows than in three-wave cows (16.7±0.30 vs. 18.40±0.50 d; P&lt;0.05). In addition, the day of peak progesterone occurred earlier in two-wave cows (13.50±0.40 vs. 16.30±0.70 d; P&lt;0.05).

Prostaglandin Synthesis, Metabolism, and Signaling Potential in the Rhesus Macaque Corpus Luteum throughout the Luteal Phase of the Menstrual Cycle

Prostaglandins in the corpus luteum (CL) reportedly serve as luteotropic and luteolytic agents. Based mainly on studies conducted in domesticated animals and rodents, prostaglandin E2 (PGE2) is generally considered a luteotropic factor, whereas uterine-derived prostaglandin F2alpha (PGF2alpha) initiates luteolysis. However, the role of prostaglandins in regulating primate luteal structure-function is poorly understood. Therefore, a comprehensive analysis of individual mRNA or proteins that are involved in PGE2 and PGF2alpha biosynthesis, metabolism, and signaling was performed using CL obtained at distinct stages of the luteal life span during the menstrual cycle in rhesus monkeys. Peak levels of proteins involved in PGE2 synthesis (prostaglandin-endoperoxide synthase 2, microsomal PGE2 synthase-1) and signaling (PGE2 receptor 3) occurred during periods corresponding to development and maintenance of the primate CL. Immunohistochemistry studies indicated that large luteal cells express PGE2 synthesizing and signaling proteins. Expression of PGE2 synthesizing and signaling proteins significantly decreased preceding the period of functional regression of the CL, which also coincided with increasing levels of PGF2alpha receptor protein expression within the large luteal cells. Moreover, significant levels of mRNA expression for several aldoketo reductase family members that synthesize PGF2alpha from other prostaglandins were observed throughout the rhesus macaque luteal phase, thus supporting the possibility of intraluteal PGF2alpha production. Collectively, our results indicate that there may be intraluteal synthesis and signaling of PGE2 during development and maintenance of the primate CL, followed by a shift to intraluteal PGF2alpha synthesis and signaling as the CL nears the time of luteolysis.

Effect of progesterone and/or estradiol treatments prior to induction of ovulation on subsequent luteal lifespan in anestrous Nelore cows

Three experiments evaluated effects of estradiol (E 2) and/or progesterone (P 4) prior to induction of ovulation with GnRH on subsequent luteal lifespan in anestrous Nelore cows. In Experiment 1, cows (25–65 days post-partum [DPP]; n = 114) were assigned randomly to receive a 6-day treatment with an intravaginal P 4 device (CIDR ®) and/or 1 mg i.m. injection of 17β-E 2 (4 groups; 2 × 2 factorial design) prior to ovulation. Blood samples were collected on days 0, 5, 7, 9, 12, 15 and 19 for evaluation of luteal function. Pre-treatment with P 4 reduced occurrence of premature luteolysis (PL; 79.2% in non-treated vs. 13.5% in treated cows; P < 0.01), but there was no effect of treatment with 17β-E 2 on percentage of PL. In Experiment 2, cows (30–40 DPP; n = 35) were assigned randomly to receive either 0.5 mL i.m. injection of cottonseed oil (placebo) or 1 mg i.m. injection of E 2 cypionate. Blood samples were collected on days 0, 5, 9 and 15 for evaluation of luteal function. Incidence of PL (83.0% in Control Group vs. 75.0% in ECP Group; P > 0.1) and mean serum P 4 did not differ between treatment groups. In Experiment 3, cows (30–60 DPP; n = 109) were randomly assigned to receive either a 6-day (6-d Group) or a 3-day (3-d Group) treatment with CIDR ®. Blood samples were collected on days 0, 5, 7 and 9 for luteal function evaluation. Incidence of PL (5.5% in 6-day vs. 5.5% in 3-day groups; P > 0.1) and mean serum P 4 did not differ between treatment groups. In conclusion, both 3- and 6-day treatments with P 4 prior to induction of ovulation in anestrous Nelore cows increased percentage of normal luteal lifespan, while administration of 1 mg of 17β-E 2 or E 2 cypionate failed to prevent occurrence of PL.

Luteal lifespan and fertility after estrus synchronization in goats

The present experiment aims to examine the efficiency of estrus synchronization using progesterone and equine chorionic gonadotrophin (eCG) and to look at luteal function. During the non-breeding and breeding season, 5 adult female Korean native goats were injected intramuscularly with 2.5 ml of physiological saline as the control. A progesterone impregnated intravaginal sponge was then kept in the same goats for 10 days followed, after a week, by an intramuscular injection of 500 IU eCG. Five adult female Nubian goats were mated with a fertile buck during the non-breeding season. During the non-breeding season 2 of the 5 goats showed a normal estrous cycle (ranging from 18 to 21 days) and 3 a short estrous cycle (ranging from 3 to 6 days). During the breeding season the equivalent figures were 1 and 2. The major axes of the corpus luteum (CL) were measured by means of calipers built into the ultrasonography system, and the concentrations of plasma progesterone (P4) were determined by double antibody radioimmunoassay. The mean major axes of the CL in goats showing the short cycle (6.1 ± 0.5 mm) was significantly smaller than in those showing the normal cycle (8.9 ± 0.5 mm; p < 0.01) and also the value of P4 in goats showing the short cycle (4.2 ± 2.1 ng/ml) was significantly lower than for those showing the normal cycle (10.3 ± 4.3 ng/ml; p < 0.05) at day 3 following ovulation. Three out of 5 Nubian goats became pregnant but only one goat carried to full term. The present experiment indicated that a combination of progesterone and eCG was effective in inducing estrus, although it resulted in a high incidence of short luteal lifespan. The low kidding rate and high incidence of embryonic loss may be due to the instability of the luteal lifespan.