Luteal blood flow measured by Doppler ultrasonography during the first three weeks after artificial insemination in pregnant and non-pregnant Bos indicus dairy cows.

The objective of this study was to determine if there are differences in luteal size (LS), progesterone (P4), and luteal blood flow (LBF) between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after insemination, and whether these parameters are related to each other. Lactating cows (n = 13) of mixed parity with a body weight of 430 ± 18 kg (mean ± SD), showing regular estrous cycle were used in the study. All cows were artificially inseminated and were classified as pregnant (embryonic heartbeat on day 30; n = 8) or non-pregnant (inter-estrus interval 17 to 21 days, n = 5). In order to compare the LS and LBF after artificial insemination, B-mode and color Doppler ultrasonography of ovaries were performed on days 4, 5, 6, 7 (first week), 8, 10, 12, 14, (second week), and 16, 17, 18, 19, 20, 21 (third week) in pregnant and non-pregnant cows. Results revealed that the mean LBF was consistently higher (P < 0.05) during days 7 through 21 in pregnant cows than in non-pregnant cows. The mean LS was higher (P < 0.05) on days 6 and 7, and from day 17 onwards, and the mean concentration of P4 was higher (P < 0.05) on days 19, 20, and 21 in pregnant cows. In conclusion, LBF is a more sensitive parameter than LS and P4 for detection of differences in luteal function between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after AI.

Vascular and morphological features of the corpus luteum 12 to 20 days after timed artificial insemination in dairy cattle.

Our objective was to retrospectively compare pregnant versus nonpregnant cattle in terms of vascular and morphometric changes in corpora lutea between d 12 and 20 following timed artificial insemination (TAI). Crossbred (Gir × Holstein) lactating dairy cows (n = 136) and heifers (n = 111) were bred after synchronizing ovulations using an estradiol plus progesterone (P4)-based protocol. Corpus luteum (CL) characteristics (area, echotexture, blood flow) were recorded at 48-h intervals from d 12 to 20 following TAI using an ultrasound equipped with color Doppler. Blood samples were collected to determine CL function (plasma P4). Pregnancy diagnosis was performed at d 30. Quantitative assessment of colored pixels within the CL was performed using ImageJ software (National Institutes of Health, Bethesda, MD) and echotexture was quantified using custom software. Continuous variables such as luteal tissue area (LTA), CL blood flow (CLBF), adjusted CLBF (ratio LTA:CLBF), mean pixel value (MPV), pixel heterogeneity (HETER), and plasma P4 were analyzed retrospectively as repeated measures (d 12 to 20) in pregnant versus nonpregnant females using PROC MIXED (SAS Institute Inc., Cary, NC). Main effects were pregnancy status, day of cycle, and their interaction. Further analyses used only data from d 16, because this was the earliest time point of deviation between CLBF of pregnant and nonpregnant animals. We created quartiles for each variable and calculated the risk of pregnancy within quartile. Differences were determined using the chi-squared test. Plasma P4 was significantly higher in prospective pregnant versus nonpregnant cattle on d 18 and 20, whereas LTA differed only on d 20. On d 16, CLBF and adjusted CLBF diverged between pregnant and nonpregnant, followed by a progressive reduction in the latter until d 20. Mean pixel value was not affected by pregnancy status, but HETER was lower on d 20 in pregnant than in nonpregnant cattle. Likelihood of pregnancy increased from quartile (Q)1 (lowest values) to Q4 (highest) of CLBF (Q4 vs. Q1, odds ratio = 32.8, 95% confidence interval: 9.6 to 112.1) and adjusted CLBF [Q4 vs. Q1, odds ratio = 25.4, 95% confidence interval: 8.1 to 80.4), whereas a lower risk of pregnancy was observed only for animals within Q1 of plasma P4 [Q4 vs. Q1, odds ratio = 3.1, 95% confidence interval: 1.3 to 7.2). Day 16 quartiles of LTA, MPV, and HETER did not affect odds of pregnancy. In conclusion, we identified distinct CLBF patterns as early as 16 d after TAI and confirmed that CL function is lost by a reduction in blood flow, which precedes physical regression.

Spatial relationships of ovarian follicles and luteal structures in dairy cows subjected to ovulation synchronization: Progesterone and risks for luteolysis, ovulation, and pregnancy.

Objectives were to determine relative ovary location of follicles, GnRH-induced corpora lutea (CL), and older CL present in ovaries as part of ovulation synchronization and their associations with progesterone concentration and risk for luteolysis, ovulation, and pregnancy. Cows were exposed to a timed artificial insemination (AI) program [GnRH-1-7 d-PGF2α (1 dose or 2 doses 24 h apart)-56 h after first or only dose of PGF2α-GnRH-2-16 h-timed AI at 72 ± 3 d in milk]. Blood was collected to assess progesterone when ovarian structures were mapped in 694 cows before GnRH-1 and before and 48 h after PGF2α and, in a subset of cows, size of CL (n = 599) and progesterone (n = 380) at 6 d after AI. Dominant follicles and CL in single-ovulating cows were detected more often in right than left ovaries (follicles before GnRH-1: 60.6% right and GnRH-2: 61.2% right; and CL before GnRH-1: 58.6% right and GnRH-2: 66.4% right). Dominant follicles in single-ovulating cows before GnRH-1 tended to be ipsilateral to the CL more often than contralateral (54.8 vs. 45.2%) with co-dominant follicles identified in both ovaries (19.3%). In response to GnRH-1 or GnRH-2, more left-ovary follicles ovulated contralateral to CL (left to right, 54.7%; right to left, 34.7%) than right-ovary follicles, but fewer left-ovary follicles ovulated ipsilateral to CL (left to left: 45.3%) than right-ovary follicles ovulated ipsilateral (right to right: 65.3%). Preovulatory follicles in single-ovulating cows before PGF2α tended to be detected more often ipsilateral than contralateral to CL induced by GnRH-1 (younger CL; 56.5 vs. 43.6%), but were of equal frequency ipsilateral or contralateral to older CL present before GnRH-1. Luteolytic risk was less in cows bearing co-dominant follicles in both ovaries compared with those in either right or left ovaries. Luteolytic risk in single-ovulating cows did not differ between ovaries. Luteolytic risk was greater for cows bearing older CL (86.5%) than for cows bearing younger GnRH-1-induced CL (65.3%) or both (79.6%). Pregnancy risk at 60 d after AI was or tended to be greater in cows having both CL types compared with either younger or older CL, respectively, partly because of greater embryonic loss in the latter 2 cases. More female calves tended to be carried in right horns when conception occurred after first service, whereas the opposite greater female frequency occurred in left horns after repeat services. Right-ovary dominance is evident before and after GnRH treatment.

Ovarian characteristics in sheep with multiple fecundity genes.

Ewes heterozygous for combinations of the Inverdale (FecXI; I+), Booroola (FecB; B+) and Woodlands (FecX2W; W+) mutations have ovulation rates higher than each mutation separately. The aims of the experiments described herein were to examine the ovarian phenotypes in I+B+ and I+B+W+ ewes and to compare these with the appropriate ++ (controls), I+ and BB animals available for this study. The mean ± s.e.m. ovulation rates in the ++ (n = 23), I+ (10), I+B+ (7), I+B+W+ (10) and BB (3) animals were 1.8 ± 0.1, 2.5 ± 0.2, 6.6 ± 1.0, 9.6 ± 0.9 and 9.7 ± 0.9 respectively. The maximum number of granulosa cells per follicle in the ++ and I+ genotypes was accumulated after exceeding 5 mm diameter, whereas in I+B+, I+B+W+ and BB animals, this was achieved when follicles reached >2-3 mm. The number of putative preovulatory follicles, as assessed from those with LH-responsive granulosa cells, 24 h after the induction of luteolysis, was higher (P < 0.01) in the I+B+ and I+B+W+ compared to the ++ and I+ genotypes. The median follicular diameters of these follicles in the ++, I+, I+B+, I+B+W+ and BB genotypes were 6, 5, 3, 3 and 3 mm respectively. The total number of granulosa cells in the putative preovulatory follicles when added together, and total mass of luteal tissue, did not differ between the genotypes. Thus, despite large ovulation rate differences between animals with one or more fecundity genes, the total cell compositions over all preovulatory follicles and corpora lutea, when added together, are similar to that from the one or two such follicles in the wild types.

Normal Developmental and Estrous Cycle-dependent Histological Features of the Female Reproductive Organs in Microminipigs.

The microminipig has become an increasingly attractive animal model for various experimental practices because of its manageable size; however, studies of the histological features of the female reproductive organs in microminipigs are limited. The present study investigates the sexual development of the reproductive organs and the cyclical changes during the estrous cycle in female microminipigs. The ovaries, oviducts, uteri, and vaginal tissues from 33 animals aged 0 to 26 months were utilized in this study. By evaluating the large tertiary follicles, corpora lutea, and the regressing corpora lutea, we estimated that female microminipigs reached puberty at approximately 5 months of age and sexual maturity at 8 months of age. The appearance of the follicles and corpora lutea in the ovaries, as well as the epithelium in other reproductive organs, was synchronized with each phase of the estrous cycle and was identical to that in common domestic pigs. In addition, several spontaneous findings were observed, including mesonephric duct remnants adjacent to oviducts and mineralization in ovaries. Understanding the normal histology of the reproductive organs in microminipigs is crucial for advancing pathological evaluations for future toxicological studies.

The effect of presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously, 7 d before Ovsynch, compared with Presynch-10/Ovsynch on luteal function and first-service pregnancies per artificial insemination.

Pregnancy per artificial insemination (P/AI) following Ovsynch is optimized when cows ovulate to the first GnRH of Ovsynch. Fertility programs are designed to presynchronize cows to d 6 or 7 of the estrous cycle to increase the chances of ovulation of a first-wave dominant follicle to the first GnRH of Ovsynch. The hypothesis of this experiment was that simplification of a presynchronization program through the combination of PGF2α and GnRH on the same day, 7 d before Ovsynch, would allow for similar P/AI compared with Presynch-10. Lactating dairy cows (n = 432) 41 to 47 d in milk (DIM) were randomly assigned to 2 treatments within parities for first service. Control cows received Presynch-10/Ovsynch consisting of the following: PGF2α-14 d-PGF2α-10 d-GnRH-7 d-PGF2α-56 h-GnRH-16 h-AI. Treated cows received PGF2α and GnRH-7 d-GnRH-7 d-PGF2α-56 h-GnRH-16 h-AI. All cows received a supplemental injection of PGF2α 24 h after the PGF2α of Ovsynch to enhance complete luteolysis. All cows received timed AI between 75 and 81 DIM. Blood was collected to assess circulating concentrations of progesterone (P4), and the number and size of corpora lutea (CL) were recorded using ultrasonography on day of PGF2α of Ovsynch. The administration of PGF2α simultaneously with GnRH and 7 d before Ovsynch (PG+G) had similar P/AI at 28 (46 vs. 48%), 35 (43 vs. 43%), 49 (39 vs. 39%), and 77 d post-AI (38 vs. 39%) compared with Presynch-10. No differences were observed in P/AI in primiparous versus multiparous cows at 28 (52 vs. 45%), 35 (48 vs. 41%), 49 (45 vs. 37%), and 77 d post-AI (43 vs. 36%). No difference existed between treatments in percentage of cows with functional CL at PGF2α of Ovsynch, total luteal area (mm2), or serum concentrations of P4 at time of PGF2α of Ovsynch, regardless of parity. Number of CL had a tendency to be greater for multiparous PG+G vs. Presynch-10 cows (2.34 ± 0.09 vs. 2.15 ± 0.08) but not in primiparous cows (1.95 ± 0.10 vs. 1.98 ± 0.11). In summary, administering both PGF2α and GnRH on the same day, 7 d before the start of Ovsynch, appears to be a simple and effective alternative to Presynch-10 Ovsynch.

Pre-Selection Test to Identify High Responder Donor Goats.

The aim of the study was to evaluate the feasibility of pre-selection of high or low responder does prior to the superovulatory protocols. Twenty Saanen does received 800 IU of equine chorionic gonadotropin (eCG) at the end of long-term progestogen treatment. Fourteen days later, a second progestogen protocol associated with a multiple-dose follicle stimulation hormone (FSH) treatment (5 IU/kg of FSH, in six decreasing doses between days 4 to 6 of the protocol) was administered. Transrectal ultrasound was used to assess the follicular status at the beginning of superovulatory treatments, at the oestrous onset and on the seventh day of the oestrous cycle for counting corpora lutea (CL). A significant lower number of CL was obtained in eCG-treated in comparision with FSH-treated does (p < 0.05). A quartic regression was able to explain the relationship between the number of CL in response to both treatments (r(2) =0.50; p < 0.05). Seventy per cent (14 of 20) of does maintained the same ovulatory response (high or low) after treatments. The Kappa (κ = 0.40; p < 0.05) and Spearman (rs = 0.39; p = 0.08) coefficients were able to show a relationship between treatments. Regarding the follicular status, there is a significant relationship between the number of small follicles (r = 0.71; r(2) =0.47; p < 0.01) and total follicles (r = 0.60; p < 0.01) at eCG and first FSH dose with the number of CL. Moreover, it was found a negative relationship between the presence of large follicles and the number of CL in response to eCG treatment (r = -0.44; p < 0.05), but not from FSH (p > 0.05). In conclusion, the screening test with eCG has the potential to identify Saanen does that will better respond to the superovulatory protocol with FSH. In addition, it highlighted the importance of an ultrasound evaluation prior to the beginning of superovulatory treatments with FSH to characterize the follicular status and identify the potential donors of high ovulatory response in MOET programmes in goats.

Exposure to di(2-ethylhexyl) phthalate inhibits luteal function via dysregulation of CD31 and prostaglandin F2alpha in pregnant mice.

BACKGROUND: Di(2-ethylhexyl) phthalate (DEHP) exposure reduces embryo implantations, increases embryonic loss, and decreases fetal body weights. However, whether it is associated with the alteration of luteal function remains unknown. Thus, our aim in this study was to explore the effect and mechanism of DEHP on luteal function in pregnant mice in vivo. METHODS: Mice were administered DEHP by gavage at 125, 250, 500 mg/kg/day from gestational days (GD) 1 to 9 or 13. Levels of serum progesterone and estradiol were measured by radioimmunoassay. The numbers and sizes of corpora lutea were calculated by ovarian histomorphology. Steroidogenic enzymes were assessed by qRT-PCR. CD31 protein was detected by immunocytochemistry, and prostaglandin F2alpha (PGF2alpha) levels were evaluated by enzyme immunoassay. RESULTS: Treatment with DEHP significantly inhibited progesterone secretion in pregnant mice in a dose-dependent manner but did not inhibit estradiol production on GD 9 and 13. Treatment also showed concomitant decreases in transcript levels for key steroidogenic enzymes (CYP11A, 3ß-HSD, and StAR) on GD 13. Furthermore, DEHP administration significantly reduced the numbers and sizes of corpora lutea on GD 13. No significant changes in the ratio of ovary weight vs. body weight were observed between the control group and treated animals on GD 9 and 13. In addition, treatment with DEHP significantly inhibited CD31 expression of corpora lutea, whereas plasma PGF2alpha levels in DEHP treatment groups were significantly higher compared with the control groups on GD 9 and 13. CONCLUSIONS: The results show DEHP significantly inhibits luteal function of pregnant mice in vivo, with a mechanism that seems to involve the down-regulation of progesterone and steroidogenic enzymes message RNA, the decrease in CD31 expression, and the increase in PGF2alpha secretion.

Ovarian function in captive owl monkeys (Aotus nancymaae and A. vociferans).

BACKGROUND: Ovarian function and morphology of owl monkeys (Aotus spp.) is inadequately known, but it is essential to describe this organ in detail to better understand reproductive patterns of the species. METHODS: This study reports the anatomical and histological features of the ovaries from 41 owl monkeys, considering their reproductive status and parity. RESULTS: The corpora lutea (CL) and reproductive state had a significant effect on ovarian volume. Follicles were observed in both pregnant and non-pregnant subjects. All females in the luteal phase and pregnant females showed at least one cyclic or pregnancy CL. Mean ovulation rate was 1.89 CL/female, and the mean ovum mortality was 37.5%. There were no significant differences in ovarian volume related to parity. CONCLUSIONS: These results contribute to the scarce information available on the reproductive biology of owl monkeys and can aid in the development of biotechnologies involved in assisted reproduction.

Effect of three pFSH doses on superovulation and embryo quality in goats during two breeding seasons in north-eastern mexico.

The aim of this research was to evaluate the effectiveness of three pFSH doses (80 mg; 145 mg and 215 mg) on ovarian response and on quantity and quality of transferable embryos of goats during the breeding and the non-breeding seasons. Ovary structures were exposed (laparatomy under general anaesthesia) and numbers of follicles and corpora lutea were registered. Surgical embryo flushing was conducted to count and classify embryos. There were more follicles (3.4 ± 1.1) in does administered 80 mg of pFSH (p < 0.05) than in goats administered 145 mg of pFSH (2.2 ± 1.1) and 215 mg of pFSH (0.9 ± 0.6). Numbers of corpora lutea, blastocysts, and recovered and transferable embryos of goats administered 145 mg pFSH (13.4 ± 3.7, 2.42 ± 1.0, 3.4 ± 1.2 and 3.2 ± 1.1, respectively) and those of goats administered 215 mg pFSH (11.6 ± 2.6, 3.2 ± 0.9, 5.7 ± 1.5, and 5.6 ± 1.5) were greater (p < 0.05) than values obtained from goats administered 80 mg pFSH (4.0 ± 1.5, 0.5 ± 0.3, 1.0 ± 0.5, and 0.8 ± 0.5). Numbers of morula of does administered 80 and 145 mg pFSH (0.4 ± 0.4 and 0.8 ± 0.3) were lower (p < 0.05) than those obtained from animals treated with 215 mg pFSH (2.4 ± 0.9). There was no effect of season upon the analyzed variables. In conclusion, under the prevalent conditions in north-eastern Mexico, administration of 145 or 215 mg pFSH in a decreasing dose schedule over 3.5 days to bred goats provided a satisfactory superovulatory result.

Global gene expression in the bovine corpus luteum is altered after stimulatory and superovulatory treatments.

Equine chorionic gonadotrophin (eCG) has been widely used in superovulation and artificial insemination programmes and usually promotes an increase in corpus luteum (CL) volume and stimulates progesterone production. Therefore, to identify eCG-regulated genes in the bovine CL, the transcriptome was evaluated by microarray analysis and the expression of selected genes was validated by qPCR and western blot. Eighteen Nelore crossbred cows were divided into control (n=5), stimulated (n=6) and superovulated groups (n=7). Ovulation was synchronised using a progesterone device-based protocol. Stimulated animals received 400 IU of eCG at device removal and superovulated animals received 2000 IU of eCG 4 days prior. Corpora lutea were collected 7 days after gonadotrophin-releasing hormone administration. Overall, 242 transcripts were upregulated and 111 transcripts were downregulated in stimulated cows (P ≤ 0.05) and 111 were upregulated and 113 downregulated in superovulated cows compared to the control animals (1.5-fold, P ≤ 0.05). Among the differentially expressed genes, many were involved in lipid biosynthesis and progesterone production, such as PPARG, STAR, prolactin receptors and follistatin. In conclusion, eCG modulates gene expression differently depending on the treatment, i.e. stimulatory or superovulatory. Our data contribute to the understanding of the pathways involved in increased progesterone levels observed after eCG treatment.

A superovulation protocol for the spiny mouse (Acomys cahirinus).

This study aimed to develop a superovulation protocol for the spiny mouse (Acomys cahirinus). The spiny mouse is a desert-adapted rodent species, with a long oestrus cycle (11 days) compared with rat and mouse, and gives birth to few (mean litter size is 3) precocial offspring after a relatively long gestation (39 days). We successfully optimised a superovulation protocol that elicited a 5-fold increase in the normal ovulation rate of this species. To induce superovulation in the spiny mouse 2 injections of equine chorionic gonadotrophin (eCG, 10 IU each), 9h apart, were required, followed by 20 IU of human chorionic gonadotrophin (hCG). This protocol was successful in 100% of females trialed and at 33 h post-hCG an average of 14.7 ± 1.5, 1-2 cell embryos were recovered. Histological analysis of ovaries following superovulation revealed large corpus lutea and post-ovulatory follicles occupying a large part of the ovary. Ovulation commenced 6-12 h after the hCG injection and continued until 24-33 h post-hCG as indicated by both histological analysis of ovaries and the presence of oocytes/embryos in the oviduct. This superovulation protocol will facilitate the development of an in vitro culture system for spiny mouse embryos.

Prostaglandin F2α and gonadotropin-releasing hormone administration improve progesterone status, luteal number, and proportion of ovular and anovular dairy cows with corpora lutea before a timed artificial insemination program.

The objective of this research was to increase the proportion of cows with at least 1 functional corpus luteum (CL) and elevated progesterone at the onset of the timed artificial insemination (TAI) protocol. Postpartum Holstein cows in one herd were stratified by lactation number at calving (September 2009 through August 2010) and assigned randomly to 2 treatments: 1) Presynch-10 (n=105): two 25-mg injections of PGF(2α) (PG) 14 d apart (Presynch); and 2) PG-3-G (n=105): one 25-mg injection of PG 3 d before 100-µg GnRH (Pre-GnRH) injection, with the PG injection administered at the same time as the second PG injection in the Presynch-10 treatment. Cows were enrolled in a TAI protocol [Ovsynch; injection of GnRH 7 d before (GnRH-1) and 56 h after (GnRH-2) PG injection with AI 16 to 18 h after GnRH-2] 10 d after the second or only PG injection. Blood samples for progesterone or estradiol analyses were collected on median days in milk (DIM): 36, 39, 50, 53 (Pre-GnRH), 60 (GnRH-1), 67 (PG), 69 (GnRH-2), and 70 (TAI). Ovarian structures were measured by ultrasonography on median DIM 53, 60, 67, 69, and 6 d post-TAI to determine follicle diameters, ovulation response to GnRH, or both. Although progesterone concentration did not differ between treatments before Pre-GnRH injection, the proportion of cows with at least 1 CL tended to be greater for PG-3-G than Presynch-10 cows, and more PG-3-G cows ovulated after Pre-GnRH injection than ovulated spontaneously in Presynch-10. Further, the diameter of follicles that ovulated tended to be smaller in PG-3-G than in Presynch-10 cows after Pre-GnRH injection. At GnRH-1, the proportion of cows with progesterone ≥1 ng/mL, the number of CL per cow, and the proportion of cows with at least 1 CL were greater for PG-3-G than Presynch-10. Neither follicle diameter nor percentage of cows ovulating after GnRH-1 differed between treatments. At PG injection during the week of TAI, progesterone concentration and the proportion of cows with progesterone ≥1 ng/mL tended to be greater for PG-3-G than Presynch-10, and PG-3-G had more CL per cow than Presynch-10. No ovarian characteristics differed between treatments after GnRH-2, including progesterone concentration, number of CL per cow, and total luteal volume 7 d after GnRH-2. Many of the previous ovarian traits were improved in both ovular and anovular cows after PG-3-G compared with Presynch-10. Pregnancies per AI at d 32 and 60 were only numerically greater for PG-3-G than for Presynch-10 cows, largely because of differences detected during months without heat stress. We concluded that the PG-3-G treatment increased ovulation rate and luteal function 7 d before the onset of Ovsynch, resulting in improved follicular synchrony and predisposing potentially greater pregnancies per AI in lactating dairy cows.

Effects of endocannabinoid 1 and 2 (CB1; CB2) receptor agonists on luteal weight, circulating progesterone, luteal mRNA for luteinizing hormone (LH) receptors, and luteal unoccupied and occupied receptors for LH in vivo in ewes.

Thirty to forty percent of ruminant pregnancies are lost during the first third of gestation due to inadequate progesterone secretion. During the estrous cycle, luteinizing hormone (LH) regulates progesterone secretion by small luteal cells (SLC). Loss of luteal progesterone secretion during the estrous cycle is increased via uterine secretion of prostaglandin F(2α) (PGF(2α)) starting on days 12-13 post-estrus in ewes with up to 4-6 pulses per day. Prostaglandin F(2α) is synthesized from arachidonic acid, which is released from phospholipids by phospholipase A2. Endocannabinoids are also derived from phospholipids and are associated with infertility. Endocannabinoid-induced infertility has been postulated to occur primarily via negative effects on implantation. Cannabinoid (CB) type 1 (CB1) or type 2 (CB2) receptor agonists and an inhibitor of the enzyme fatty acid amide hydrolase, which catabolizes endocannabinoids, decreased luteal progesterone, prostaglandin E (PGE), and prostaglandin F(2α) (PGF(2α)) secretion by the bovine corpus luteum in vitro by 30 percent. The objective of the experiment described herein was to determine whether CB1 or CB2 receptor agonists given in vivo affect circulating progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors during the estrous cycle of ewes. Treatments were: Vehicle, Methanandamide (CB1 agonist; METH), or 1-(4-chlorobenzoyl)-5-methoxy-1H-indole-3-acetic acid morpholineamide (CB2 agonist; IMMA). Ewes received randomized treatments on day 10 post-estrus. A single treatment (500 µg; N=5/treatment group) in a volume of 1 ml was given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. Jugular venous blood was collected at 0 h and every 6-48 h for the analysis of progesterone by radioimmunoassay (RIA). Corpora lutea were collected at 48 h, weighed, bisected, and frozen in liquid nitrogen until analysis of unoccupied and occupied LH receptors and mRNA for LH receptors. Profiles of jugular venous progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors were decreased (P≤0.05) by CB1 or CB2 receptor agonists when compared to Vehicle controls. Progesterone in 80 percent of CB1 or CB2 receptor agonist-treated ewes was decreased (P≤0.05) below 1 ng/ml by 48 h post-treatment. It is concluded that the stimulation of either CB1 or CB2 receptors in vivo affected negatively luteal progesterone secretion by decreasing luteal mRNA for LH receptors and also decreasing occupied and unoccupied receptors for LH on luteal membranes. The corpus luteum may be an important site for endocannabinoids to decrease fertility as well as negatively affect implantation, since progesterone is required for implantation.

In vivo intra-luteal implants of prostaglandin (PG) E(1) or E(2) (PGE(1), PGE(2)) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteal luteinizing hormone (LH) receptor, and occupied and unoccupied luteal receptors for LH.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every four hours inhibited luteolysis in ewes. However, estradiol-17ß or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in heifers, but infusion of estradiol+PGE(2) inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE(1) or PGE(2) prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE(1) or PGE(2) on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P>0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13-19 were lower (P<0.05) than those treated with intra-luteal implants containing PGE(1) or PGE(2). Day-13 Angus luteal weights were heavier (P<0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P>0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE(1) or PGE(2). Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE(1) or PGE(2) differed (P<0.05) from controls, but profiles of progesterone did not differ (P>0.05) between breeds or between cows treated with intra-luteal implants containing PGE(1) or PGE(2). Intra-luteal implants containing PGE(1) or PGE(2) prevented (P<0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE(1) or PGE(2) alone delays luteolysis regardless of breed. We also conclude that either PGE(1) or PGE(2) prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue.

Vascular endothelial growth factor is essential for corpus luteum angiogenesis.

The development and endocrine function of the ovarian corpus luteum (CL) are dependent on the growth of new capillary vessels. Although several molecules have been implicated as mediators of CL angiogenesis, at present there is no direct evidence for the involvement of any. Here we report the unexpected finding that treatment with truncated soluble Flt-1 receptors, which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in virtually complete suppression of CL angiogenesis in a rat model of hormonally induced ovulation. This effect was associated with inhibition of CL development and progesterone release. Failure of maturation of the endometrium was also observed. Areas of ischemic necrosis were demonstrated in the corpora lutea (CLs) of treated animals. However, no effect on the preexisting ovarian vasculature was observed. These findings demonstrate that, in spite of the redundancy of potential mediators, VEGF is essential for CL angiogenesis. Furthermore, they have implications for the control of fertility and the treatment of ovarian disorders characterized by hypervascularity and hyperplasia.

Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow.

We investigated changes in peripheral concentrations of inhibin A, total inhibin, steroids, and gonadotropins throughout the intact estrous cycle of the sow in relation to ovarian changes determined by daily transrectal ultrasonography. All visible follicles of 3 mm or more in diameter were classified as small (> or =3 and <6 mm) or large (> or =6 mm). Follicular recruitment was identified in two periods of the cycle: one from the late luteal to the follicular phase, characterized by an increase in the number of small follicles followed by the appearance of large follicles; and another during the early luteal phase, consisting only of increased numbers of small follicles. Plasma concentrations of inhibin A increased (P<0.05), coinciding with the two periods of follicle emergence. Estradiol (E(2)) levels increased (P<0.05) during the follicular phase, but not during the early luteal phase. An inverse relationship (P<0.01) between the patterns of inhibin and FSH concentrations was noted around the two periods of follicle emergence, but there was no relationship (P> or =0.1) between the patterns of plasma E(2) and FSH during the early luteal phase. In conclusion, measurement of plasma inhibin A levels combined with ultrasonographic examination of the ovaries revealed two periods of synchronous follicular growth during the sow's estrous cycle. The results strongly suggest that inhibin A functions as a negative feedback regulator of FSH secretion throughout the estrous cycle, whereas E(2) appears to influence FSH secretion only during the follicular phase.

Effects of prostaglandin E and F receptor agonists in vivo on luteal function in ewes.

Loss of progesterone secretion at the end of the estrous cycle is via uterine PGF(2alpha) secretion; however, uterine PGF(2alpha) is not decreased during early pregnancy in ewes to prevent luteolysis. Instead the embryo imparts resistance to PGF(2alpha)-induced luteolysis, which is via the 2-fold increase in prostaglandins E(1) and E(2) (PGE(1), PGE(2); PGE) in the endometrium during early pregnancy. Chronic intrauterine infusion of PGE(1) or PGE(2) prevents spontaneous or an estradiol-17beta, IUD, or PGF(2alpha)-induced luteolysis. Four PGE receptor subtypes (EP(1), EP(2), EP(3), and EP(4)) and an FP receptor specific for PGF(2alpha) have been identified. The objective of this experiment was to determine the effects of EP(1), EP(2), EP(3), or FP receptor agonists in vivo on luteal mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone in ewes. Ewes received a single treatment of 17-phenyl-tri-Nor-PGE(2) (EP(1), EP(3)), butaprost (EP(2)), 19-(R)-OH-PGE(2) (EP(2)), sulprostone (EP(1), EP(3)), or PGF(2alpha) (FP) receptor agonists into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. 17-Phenlyl-tri-Nor-PGE(2) had no effect (P> or =0.05) on any parameter analyzed. Butaprost and 19-(R)-OH-PGE(2) increased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. Both sulprostone and PGF(2alpha) decreased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. It is concluded that both EP(3) and FP receptors may be involved in luteolysis. In addition, EP(2) receptors may mediate prevention of luteolysis via regulation of luteal mRNA for LH receptors to prevent loss of occupied and unoccupied LH receptors and therefore to sustaining luteal function.

Relationships between size, steroidogenesis and miRNA expression of the bovine corpus luteum.

In a previous study, a subset of miRNAs were identified the expression of which increases substantially during the follicle-luteal transition in cattle. Here, we investigated the functional involvement of some of these miRNAs (miR-96, miR-182, miR-132, miR-21, miR-378) by determining whether there is an association in vivo between their expression in the corpus luteum (CL), CL size and progesterone production. The two largest and two smallest CL were collected from 12 donor beef heifers on Day 7 following ovarian super-stimulation (Day 0 = 28-32 h after first standing to be mounted). Additionally, the CL and a plasma sample were collected from 29 recipient heifers on Day 15. Luteal expression of miRNAs and mRNAs, and plasma progesterone concentrations were quantified by RT-qPCR and RIA, respectively. There were no differences in the mean expression of any miRNAs examined or the steroidogenic enzymes, STAR or CYP11A1, between the largest and smallest CL in donor heifers (P > 0.1). In addition, there were no significant correlations of luteal volume or weight with any miRNA, CYP11A1 or STAR in donor heifers. However, a correlation (r ≥ 0.5, P ≤ 0.001) existed between the transcript levels of CYP11A1 and STAR in the CL, as well as between each of those and miR-182 levels. In addition, CYP11A1 abundance was moderately correlated (r ≤ 0.4, P < 0.05) with each of miR-96 and miR-378. In recipient heifers, progesterone levels were moderately correlated with luteal weight (r = 0.41, P = 0.03) but not with the expression of any miRNA, CYP11A1 or STAR (P > 0.1). Moreover, luteal CYP11A1 and STAR were correlated (r = 0.6, P ≤ 0.001) with miR-182 as well as with each other, consistent with data in donor heifers. Finally, both CYP11A1 and STAR were moderately correlated (r ≤ 0.5) with miR-132 and, in the case of STAR, with miR-378. In summary, there was no association between either luteal weight/volume or plasma progesterone concentrations and any of the miRNAs analysed in donor and recipient heifers. However, CYP11A1 and STAR transcript levels were significantly correlated with several miRNAs, most notably miR-182, as well as with each other, in luteal tissues from both donor and recipient heifers. This finding confirms results of previous in vitro studies and, importantly, provides the first in vivo evidence of a role of the miR-183-96-182 cluster in regulating luteal steroidogenesis.

Two corpora lutea seen at 6-13 weeks' gestation infers dizygosity among spontaneous same-sexed dichorionic twins.

Current ultrasound techniques can accurately determine the chorionicity of twins, but not zygosity. We previously proposed that the zygosity of spontaneously conceived twins can be determined at early ultrasound, where 2 corpora lutea infers dizygosity, and 1 implies monozygosity. Here we did a case series, comparing zygosity predicted using this method with definitive DNA genotyping of twins after birth. We retrospectively identified 14 ultrasound reports of spontaneous twin pregnancies at 6(+0 days) to 13+6 weeks' gestation, where both ovaries were seen and the number of corpora lutea documented. We visited all twin pairs, obtained buccal smears, and determined zygosity by genotyping 9 independent microsatellite markers. All 8 cases where 2 corpora lutea were seen were dizygotic pregnancies. One further case where 3 corpora lutea were seen was also dizygotic. All 3 sets of monozygotic twins had 1 corpus luteum. There were 2 cases incorrectly assigned, where 1 corpus luteum was seen in dizygotic pregnancies. We conclude if 2 corpora lutea are seen at a first trimester ultrasound of spontaneously conceived dichorionic twins, they appear to be almost certainly dizygotic. However, if 1 corpus luteum is seen in dichorionic twins, zygosity cannot be determined with certainty since it is either monozygotic, or dizygotic where a second corpus luteum has been missed.