Luteal Tissue Research Articles

Effect of combined exogenous progesterone with luteotrophic support via equine chorionic gonadotrophin (eCG) on corpus luteum development, circulating progesterone concentrations and embryo development in cattle.

The aim was to examine the effect of a single intramuscular (i.m.) injection of equine chorionic gonadotrophin (eCG) on Day 3 after oestrus on corpus luteum (CL) development, circulating progesterone and conceptus development in cross-bred beef heifers. In Experiment 1, heifers received: (1) saline, or a single i.m. injection of eCG on Day 3 at (2) 250IU (3) 500IU (4) 750IU or (5) 1000IU. Administration of eCG resulted in increased luteal tissue area and progesterone and oestradiol concentrations compared with controls. In Experiment 2, heifers received (1) a progesterone-releasing intravaginal device (PRID Delta) from Day 3 to 5 or (2) a PRID Delta from Day 3 to 5 plus a single injection of 750IU eCG on Day 3. In vitro-produced blastocysts (n=10 per recipient) were transferred on Day 7 and heifers were slaughtered on Day 14 to assess conceptus development. Administration of eCG reduced the number of short cycles (6.3% vs 31.3%) and increased mean luteal tissue weight (P=0.02). Insertion of a PRID Delta on Day 3 resulted in an elevation (P<0.05) in serum progesterone until removal on Day 5. Administration of eCG at the time of PRID Delta insertion resulted in higher progesterone levels (P<0.05) from Day 10 onwards. Conceptus dimensions were not affected. In conclusion, a single injection of eCG on Day 3 increased CL size and progesterone concentrations and, when given in conjunction with a progesterone-releasing device, appeared to reduce the number of short cycles, presumably due to its luteotrophic nature. The implications of the elevated oestradiol concentrations for embryo quality require further study.

Treatment with human chorionic gonadotrophin before ovulation increases progestin concentration in early equine pregnancies

For prevention of early conceptus loss in the horse, treatment with progestins has become common practice. In cattle, treatment with human chorionic gonadotrophin (hCG) during the early postovulatory phase stimulates endogenous progesterone synthesis, which is an important factor for maintenance of early pregnancy via stimulation of endometrial function and conceptus development. In the present study we have therefore investigated the influence of treatment with hCG either for induction of ovulation or during the early luteal phase on plasma progestin concentrations, size of the corpus luteum and size of the conceptus in early pregnant mares. We hypothesized that hCG treatment stimulates progestin secretion and conceptus development. In Experiment 1, induction of ovulation with hCG (1500IU i.v.; n=14) significantly increased progestin concentration between days 5 and 15 after ovulation compared to untreated controls (n=28; p<0.05; e.g. day 5 hCG i.v.: 17.2±1.9, control: 13.9±0.8ng/ml). A significant interaction (p<0.05) of hCG treatment with size of the conceptus between days 30 and 40 of pregnancy was detected. In Experiment 2, treatment of mares with hCG (5000IU) on day 5 after ovulation (n=12) did neither affect progestin secretion (e.g. day 8 hCG: 15.4±1.6, control: 17.6±1.2ng/ml) nor luteal tissue area (e.g. day 8 hCG: 9.0±0.7, control: 7.6±1.4cm2) compared to untreated mares (n=9). In conclusion, treatment of mares with hCG for induction of ovulation within 48h before ovulation but not on day 5 of the luteal phase stimulates progestin secretion and may enhance conceptus development via stimulation of endometrial function during early pregnancy.

Alterations in luteal production of androstenedione, testosterone, and estrone, but not estradiol, during mid- and late pregnancy in pigs: Effects of androgen deficiency

Recently, we have found that flutamide-induced androgen deficiency altered progesterone production in the porcine corpus luteum (CL) during mid- and late pregnancy. Herein, we tested whether flutamide administration subsequently influences androgen and estrogen metabolism in the CL of pregnancy. Pregnant gilts were treated with flutamide between Days 43 and 49 (GD50F), 83 and 89 (GD90F), or 101 and 107 (GD108F) of gestation. Corpora lutea (CLs) were collected from treated and nontreated (control) pigs. The concentrations of androstenedione (A4), testosterone (T), estrone (E1), and estradiol (E2) together with the levels of expression of mRNAs and proteins for cytochrome P450 17α-hydroxylase/c17-20 lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), cytochrome P450 aromatase (CYP19A1), and 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) were measured in the CL of control and flutamide-treated animals. Steroidogenic enzymes were also immunolocalized in luteal tissues. The luteal concentrations of A4 and T were higher in the GD50F (P = 0.006, P = 0.03) and GD108F (P = 0.005, P = 0.035) groups, but lower in the GD90F (P = 0.004, P = 0.014) group. The E1 level was greater only in the GD90F (P = 0.03) and GD108F (P = 0.035) groups, whereas E2 concentration was not affected by flutamide treatment. Increased luteal CYP17A1 mRNA and protein expression was found in the GD50F (P = 0.002, P = 0.03) and GD108F (P = 0.0026, P = 0.03) groups, but reduced in the GD90F (P = 0.002, P = 0.03) group. mRNA of 17β-HSD1 was upregulated in the GD50F (P = 0.0005) group, but downregulated in the GD90F (P = 0.002) and GD108F (P = 0.0005) groups. In contrast, 17β-HSD1 protein expression was higher in the GD50F and GD108F (P = 0.03) groups, but lower in the GD90F (P = 0.03) group. Both CYP19A1 mRNA and protein levels were greater in the GD90F (P = 0.001, P = 0.028) and GD108F (P = 0.005, P = 0.03) groups. Neither 17β-HSD7 mRNA nor protein level were affected by flutamide exposure. Both CYP17A1 and 17β-HSD1 were immunolocalized exclusively in small luteal cells, whereas CYP19A1 and 17β-HSD7 were found in large luteal cells of control and flutamide-treated CLs. Overall, flutamide administration led to the alterations in A4, T, and E1, but not in E2, production in the CL of pregnancy in pigs, probably because of disrupted steroidogenic enzymes expression. These changes suggest that androgens are important modulators of luteal function during pregnancy in pigs.

The effect of leptin on luteal angiogenic factors during the luteal phase of the estrous cycle in goats

Fibroblast growth factor 2 (FGF2), angiopoietin 1 (Ang1), and vascular endothelial growth factor (VEGF) are angiogenic factors implicated in the vascular development of the corpus luteum (CL). Each factor is regulated or influenced by leptin in non-ovarian tissues. Moreover, leptin and its receptor, ObRb, have been identified in luteal tissue throughout the luteal phase. Therefore, leptin is hypothesized to influence luteal vasculature through the regulation of FGF2, Ang1, and VEGF. Multiparous, cycling crossbred female goats (does) were allocated to early (n=12), mid (n=8), and late (n=11) stages of the luteal phase for CL collection. Luteal tissue was harvested and either snap frozen in liquid N2, paraffin embedded, or cultured with leptin (0, 10−12, 10−11, 10−10, 10−9, 10−8M). Tissue was analyzed for FGF2, Ang1, VEGF, ObRb, and leptin expression. Angiopoietin 1, FGF2, VEGF expression was higher (P≤0.001) in the mid-luteal stage than the early stage. Expression decreased (P≤0.001) during the late luteal stage with the exception of VEGF, which remained elevated. In contrast, leptin and ObRb were lowest (P≤0.003) during the mid-luteal stage compared to the early and late stages. All factors were detected in and/or around vessels in early stage tissue compared to mid and late stages. Leptin stimulated (P≤0.02) Ang1, FGF2, and VEGF expression only in early stage luteal cultures. Collectively, these data provide evidence that leptin may be involved in the luteal angiogenic process during the early stage of CL formation.

Macroscopic and histological characteristics of fluid-filled ovarian structures in dairy cows

The primary objective of this study was to use macroscopic and histological features of corpora lutea with a cavity and anovulatory cystic ovarian structures, present in 90 pairs of abattoir-derived dairy cow ovaries, as the basis to clarify the nomenclature of ovarian structures. Excluding morphologically normal ovarian fol-licles (antrum < 2 cm, wall < 1 mm), there were 27 fluid-filled ovarian structures. Ovulatory structures > 16 mm in diameter were designated as Group A (cavity ≤ 10 mm and wall > 10 mm) or Group B (cavity > 10 mm and wall < 10 mm). The volume of luteal tissue was less (P < 0.05) in Group B than in Group A, whereas that of a solid corpus luteum (CL) was intermediate (least square means ± SEM: 72 ± 1.92, 11.22 ± 1.57 and 5.84 ± 1.92 cm3, respectively). There was a greater proportion (P < 0.05) of small luteal cells in Group B compared to a solid CL, whereas Group A was intermediate (58.6 ± 5.3, 37.4 ± 5.3 and 44.0 ± 4.4%, respectively). Connective tissue was thicker (P < 0.05) in Group B than in Group A (295.4 ± 46.9 vs. 153.9 ± 38.2 μm). Based on the above-mentioned characteristics and differences, Groups A and B were designated as a CL with a cavity and a cystic CL, respectively. Furthermore, there were three groups of anovulatory ovarian structures. Structures in Group C were termed persistent/anovulatory follicles (overall diameter and wall thickness ≤ 20 and 1-3 mm, respectively). Finally, Groups D and E were designated as a follicle-fibrous cyst and a follicle-luteinised cyst (based on histological structure) for anovulatory structures with an overall diameter and wall thickness of ≥ 20 and ≤ 3 mm, and ≥ 20 and ≥ 3 mm, respectively.

Auto-amplification system for prostaglandin F2α in bovine corpus luteum.

The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2α production. The objective of the present study was to determine if such a PGF2α auto-amplification system exists in the bovine CL, and if so, which factors regulate it. PGF2α significantly stimulated intra-luteal PGF2α production in all luteal phases, but did not affect PGE2 production. The stimulatory effect of exogenous PGF2α on CL PGF2α production was lower at the early luteal phase. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthase (PTGS), significantly suppressed the PGF2α-stimulated PGF2α production by luteal tissue, indicating that the PGF2α in the medium was of luteal origin. Consistent with these secreted-PGF2α profiles, PGF2α receptor (PTGFR) protein expression was higher during the mid and late luteal phases than at early and developing luteal phases. Treatment of cultured bovine luteal cells obtained from the mid-luteal phase with PGF2α (1 µM) significantly increased the expressions of PTGS2, PGF synthase (PGFS), and carbonyl reductase1 (CBR1) at 24 hr post-treatment. Together, these results suggest the presence of a local auto-amplification system for PGF2α mediated by PTGS2, PGFS, and CBR1 in the bovine CL, which may play an important role in luteolysis.

Diffraction-enhanced Synchrotron Imaging of Bovine Ovaries Ex Vivo

ObjectivesThe objective of this study was to test the hypothesis that diffraction-enhanced imaging (DEI), a synchrotron x-ray imaging technique, would provide greater contrast for evaluating bovine ovaries compared with conventional diagnostic ultrasonography. Materials and MethodsBovine ovaries were evaluated ex vivo as follows: fresh without radiographic arterial contrast (n = 2), fresh with contrast (n = 1), preserved in 10% formalin without contrast (n = 2), and preserved with contrast (n = 1). Each ovary was imaged with DEI and subsequently with ultrasonography and histology. The ability to visualize and differentiate preantral and antral follicles, corpora lutea (CL), and cumulus oocyte complexes (COCs) were compared using DEI, ultrasonography, and histology. The diameter of follicles and CL were measured and compared using ultrasonography, DEI, and histology. The diameter of the smallest follicle detected was reported using each of the three imaging methods. The number of antral follicles (antral follicle count ≥2 mm) was compared between ultrasonography and DEI. ResultsDEI enabled the detection of 71% of follicles and 67% of CL that were detected ultrasonographically. However, DEI did not allow the detection of COCs and cell layers of the follicle wall that were visualized histologically. Luteal tissues were not easily distinguished using DEI, and DEI was inferior for differentiating follicles and CL compared with ultrasonography. The mean follicle diameter was similar between DEI (4.00 ± 0.35 mm, fresh with contrast; 9.62 ± 2.43 mm, fresh without contrast) and ultrasonography (3.85 ± 0.28 mm, fresh with contrast; 8.97 ± 2.60 mm, fresh without contrast) (P > .05). However, the mean follicle diameter was greater using both DEI (4.00 ± 0.35 mm) and ultrasonography (3.85 ± 0.28 mm) compared with histology (2.21 ± 0.38 mm; P = .01, fresh ovaries with contrast). The mean CL diameter was similar between DEI (11.64 ± 1.67 mm), ultrasonography (9.34 ± 0.35 mm), and histology (9.59 ± 0.36 mm) (P > .05). The mean diameter of the smallest follicle detected was similar between DEI (3.06 ± 0.45 mm) and ultrasonography (2.95 ± 0.74 mm); both DEI and ultrasonographic measurements were greater than histology (0.39 ± 0.04 mm, P < .0001). The mean antral follicle count was similar between ultrasonography (6.50 ± 0.71 mm, fresh with no contrast; 6.50 ± 2.50 mm, preserved with no contrast) and DEI (4.50 ± 0.50 mm, fresh with no contrast; 6.50 ± 0.50 mm, preserved with no contrast) (P > .05). ConclusionsThe contrast resolution of antral follicles, CL, and COCs in bovine ovaries was inferior using DEI compared with ultrasonography and histology. Alternative synchrotron techniques, such as phase-contrast computed tomography and DEI computed tomography, may prove more effective than DEI for imaging ovaries ex vivo.

Long-acting neuroleptic use for reproductive management of non-domestic ungulates using the domestic goat (Capra hircus) as a model

Fluphenazine decanoate is a long-acting phenothiazine neuroleptic that attenuates the stress response and may be useful during intensive handling for reproductive procedures in non-domestic ungulates. However, phenothiazines are also associated with elevated serum prolactin, which can suppress fertility in some species. For this study, 10 female domestic goats were used as a model for non-domestic caprids to test effects of fluphenazine decanoate on serum cortisol and reproductive cyclicity following estrus synchronization. Two identical trials were conducted during the breeding season, employing a random crossover design. First, females underwent estrus synchronization using a 14-day treatment with progesterone (330 mg; CIDR). After 7 days of CIDR exposure, the treatment group (n = 5) received fluphenazine decanoate (1.0 mg/kg IM) and controls (n = 5) received an equivalent volume of 0.9% saline IM. At CIDR withdrawal (Day 14), animals received 125 mg cloprostenol sodium to lyse any luteal tissue and synchronize estrus. Blood was collected every 2 hr from 36 hr after CIDR withdrawal until 24 hr after standing estrus, or up to 5 days to monitor stress and reproductive hormones. Serum cortisol, prolactin, luteinizing hormone (LH) and progesterone concentrations were determined by enzyme immunoassay. While treatment with fluphenazine was associated with lower cortisol concentrations compared to controls (P = 0.001), 4 of the 10 treated animals experienced elevated serum prolactin, suppression of the LH surge and inhibition of ovulation. These findings suggest that long-acting neuroleptic drugs reduce the adrenal stress response, but may interfere with reproductive responses and negatively influence breeding success.

Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas

The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 μg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.

The effects of LH on progesterone production by cell subpopulations isolated from early and late luteal phase goat corpora lutea

The objective of this study was to investigate the complementary effects of luteinizing hormone (LH) on cell subpopulations. Corpora lutea were collected on days 5 and 15 of the cycle. The luteal tissues were treated with collagenase for cell dissociation. Percoll density-gradient centrifugation was performed to separate cells into 2 distinct subpopulations. Luteal cells (5 × 104 cells/well) were cultured with and without 22(R)-hydroxycholesterol (22R-HC, 10 µg/mL) and LH (10 and 100 ng/mL) alone, and also with 22R-HC (10 µg/mL) plus LH (10 and 100 ng/mL), in a serum-free culture medium for 6 days of incubation. Untreated cells isolated from luteal tissue collected on day 15 of the estrous cycle produced more steroids than those collected on day 5. Treatment of the cells with LH resulted in a significant (P < 0.05) increase in steroid synthesis on both days 5 and 15 of the cycle. Throughout the 7 days of incubation, 22R-HC caused a significant increase in progesterone production compared to untreated controls and to the LH treatments (P < 0.01). As a result, it appears that LH is mainly effective on small luteal cells in both the early and late luteal stages in goats.

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

Physiological and practical effects of progesterone on reproduction in dairy cattle

The discovery of progesterone (P4) and elucidation of the mechanisms of P4 action have an important place in the history of endocrinology and reproduction. Circulating P4 concentration is determined by a balance between P4 production, primarily by the corpus luteum (CL), and P4 metabolism, primarily by the liver. The volume of luteal tissue and number and function of large luteal cells are primary factors determining P4 production. Rate of P4 metabolism is generally determined by liver blood flow and can be of critical importance in determining circulating P4 concentrations, particularly in dairy cattle. During timed artificial insemination (AI) protocols, elevations in P4 are achieved by increasing number of CL by creating accessory CL or by supplementation with exogenous P4. Dietary manipulations can also alter circulating P4, although practical methods to apply these techniques have not yet been reported. Elevating P4 before the timed AI generally decreases double ovulation and increases fertility to the timed AI. Near the time of AI, slight elevations in circulating P4, possibly due to inadequate luteal regression, can dramatically reduce fertility. After AI, circulating P4 is critical for embryo growth and establishment and maintenance of pregnancy. Many studies have attempted to improve fertility by elevating P4 after timed AI. Our recent meta-analysis and manipulative study indicated small fertility benefits (3% to 3.5%) mostly in primiparous cows. Thus, previous research has provided substantial insight into mechanisms regulating circulating P4 concentrations and actions. Understanding this prior research can focus future research on P4 manipulation to improve reproductive success.

Auto-paracrine action of prostaglandins E2 and F2α in equine corpus luteum

The corpus luteum (CL) is a transient endocrine organ that mainly secretes P4, a determinant hormone for implantation. In the mare, gestation depends not only on P4 action, but also on other luteotrophic factors. Prostaglandins (PG) play a critical role in several reproductive events, i.e. ovulation, luteolysis, fertilization and implantation. Prostaglandin E2 is known as a luteotrophic factor, since it stimulates in vitro P4 production by luteal steroidogenic cells in different species. Conversely, PGF2a is known as the main luteolytic factor, which is primarily secreted by the endometrium. Oxytocin (OXT) is suggested as amain inductor of pulsatile PGF2a output from the uterus during luteolysis in the mare. However, the mechanisms regulating PGs secretion and their action in the equine CL are still unknown. Thus, the aim of this study was to determine: (i) the concentration of PGs and mRNA transcription of PG synthesis enzymes in the equine CL, throughout the luteal phase; and (ii) the putative actions of PG on CL secretory function in the mid-luteal phase. In experiment 1, CLs were obtained after slaughter from early, mid-, and late CL luteal phases (n1⁄46/stage). Luteal structures were classified based on plasma P4 levels, follicle size, and the morphological appearance of the CL. Relative quantification (qPCR) of mRNA transcription from prostaglandin-endoperoxide synthase 2 (PTGS-2), synthase prostaglandin E (PGES) and F2a (PGFS) were performed. Additionally, PGE2 and PGF2a were extracted from luteal tissue and its concentration determined by ELISA method. In experiment 2, mid-CL explants (n1⁄45) were cultured for 24h with PGE2 and PGF2a (both 10-7M). Concentration of P4, PGF2a, and OXT in culture media was determined by ELISA and qPCR for 3b-hydroxysteroid dehydrogenase (3bHSD), steroidogenic acute regulatory protein (StAR), PTGS-2, PGES, PGFS, peptidyl-glycine-alpha-amidase (PGA), and OXT receptor (OXTR) were performed. In experiment 1, both PGs presented the lowest concentration level in the mid-CL, with respect to early and late CL (P<0.05), being the highest PGs concentration seen in the early CL (P<0.05). RegardingmRNA transcription, PTGS-2 and PGES presented the highest level in the early CL (P<0.01) and decreased in the subsequent stages of the luteal phase. The PGFS showed the highest expression in the mid-CL (P<0.05). In experiment 2, both PGE2 and PGF2a stimulated P4 and OXT secretion, as well as the mRNA transcription of PTGS-2, PGES, OXTR, 3bHSD (P<0.05), but not StAR and PGFS. In addition, PGE2 inhibit PGF2a secretion and increased mRNA level of PGA (P<0.05). The present results confirm that PGs are actively synthesized in the equine CL and may be involved in its auto-, paracrine regulation. Generally, PGE2 acted as a luteotrophic factor, increasing in vitro P4 secretion and also modulating OXT output in mid-CL in the mare. Further studies are needed to better understand PGs actions in CL.

Evidence for local neuroendocrine signaling in ovarian prolactin regulation

s / Journal of Equine Veterinary Science 34 (2014) 108 108 winter or during the luteal phase. PRLr were richly distributed throughout luteal tissue of both summer and winter cycling ovaries. Luteal PRLr distributionwas uniform within the CL and did not vary between luteal stages. PRLr were also present in oocytes from all follicle stages and was detected in the ovulation fossa. No other ovarian structures contained PRLr IR product.

ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression†

The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2α(PGF) for 0-4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone.

Ultrasonic Assessment of Corpora Lutea and Plasma Progesterone Levels in Early Pregnant and Non Pregnant Cows

Corpus luteum cross sectional (by ultrasonography) and plasma progesterone (by DELFIA) were estimated in early pregnant and non pregnant cows on days 14th and 20th to 23rd post insemination. On day 14th, corpus luteum sectional area was 348.43 mm2 in pregnant and 387.84mm2 in non pregnant cows. Within days 20th to 23rd, corpus luteum sectional area ranged between 342.06 and 367.90 mm2 in pregnant and between 193.85 and 270.69 mm2 in non pregnant cows. Plasma progesterone level was 2.43 ng/ml in pregnant and 2.46 ng/ml in non pregnant cows on day 14th , while during days 20th to 23rd the level ranged between 2.47 and 2,84 ng/ml in pregnant and between 0.53 and 1.17 ng/ml in non pregnant cows. Results of both luteal tissue areas as well as plasma progesterone levels were highly significantly deferent (P<0.01) between pregnant and non pregnant cows during days 20th to 23rd, but there were no significant differences on day 14th. The correlation between CL cross sectional area and plasma progesterone level was 0.4 in pregnant cows and 0.99 in non pregnant cow. It is clear, from this study, that ultrasonic assessment of corpora lutea is a viable alternative to determine plasma progesterone levels for early pregnancy diagnosis in cows.

Expression and regulation of secreted phosphoprotein 1 in the bovine corpus luteum and effects on T lymphocyte chemotaxis

Secreted phosphoprotein 1 (SPP1) in the bovine corpus luteum (CL) regulates cell function during the transitional periods of luteinization and luteal regression. The objectives were to i) characterize SPP1 expression in the CL throughout the estrous cycle, ii) determine factors that regulate SPP1 expression in luteal cells, and iii) examine the role of SPP1 in lymphocyte chemotaxis, proliferation, and function. SPP1 mRNA was greater in fully functional (d10) CL and late cycle (d18) CL compared with developing (d4) CL. Additionally, SPP1 mRNA increased within 1 h and remained elevated 4 and 8 h following induction of luteolysis with prostaglandin (PG)F2α. Expression of the SPP1 receptor, β3 integrin, was not different throughout the estrous cycle but decreased following induction of luteolysis. Expression of CD44 increased during the estrous cycle but did not change during luteal regression. In cultured luteal cells, SPP1 mRNA was upregulated by PGF2α and/or tumor necrosis factor α. Western blots revealed the presence of both full-length SPP1 and multiple cleavage products in cultured luteal cells and luteal tissue. Depletion of endogenous SPP1 did not hinder luteal cell-induced lymphocyte proliferation or lymphocyte phenotype but did inhibit lymphocyte migration toward luteal cells. Based on these data, it is concluded that SPP1 is initially activated to establish and maintain cellular interactions between steroidogenic and nonsteroidogenic cells during the development of the CL. Upon induction of luteolysis, SPP1 serves as a signaling molecule to recruit or activate immune cells to facilitate luteal regression and tissue degradation.

2-Methoxyestradiol in the human corpus luteum throughout the luteal phase and its influence on lutein cell steroidogenesis and angiogenic activity

To quantitate 2-methoxyestradiol (2-ME) in human corpus luteum (CL) of different ages and to determine the expression of cytochrome-P450-1A1 (CYP1A1) and catechol-O-methyl transferase (COMT) in CL and the action of 2-ME on P, vascular endothelial growth factor (VEGF) secretion, and luteal angiogenesis. Experimental study. University division of reproductive endocrinology. Twenty-four women of reproductive age. CL was collected from 15 women during the minilaparotomy for tubal sterilization. Granulosa lutein cells were harvested 36 hours after hCG administration in patients undergoing IVF. Levels of 2-ME were determined by high-performance liquid chromatography in CL. CYP1A1 and COMT were assessed by immunohistochemistry and Western blot. P and VEGF were measured by radioimmunoassay and ELISA. The angiogenic potential was analyzed using EA.hy926 cells. Plasma levels of E₂ decreased in the late luteal phase in association with an increase in luteal tissue of 2-ME concentrations. Concomitantly, there was a significant reduction of angiogenic activity in late CL. There was no significant variation in CYP1A1 and COMT expression in all CL. In physiological doses, 2-ME inhibited basal VEGF by granulosa lutein cells and diminished the angiogenic activity in conditioned media but did not prevent P and VEGF production stimulated by hCG. These data suggest the participation of 2-ME in physiological luteolysis by reducing angiogenesis. However, 2-ME did not prevent in vitro hCG stimulation of P biosynthesis, providing a mechanism for CL rescue in the cycle of conception.

Functional morphology of the genital organs in the wild paca (Cuniculus paca) female

Functional morphology of the genital organs is a key knowledge component for enhanced understanding of physiological patterns and for the determination of the reproductive performance in wild species. This study examines the morphology of genital organs of 133 paca females in the wild. Estimated conceptions and parturitions were mostly (83.7% and 75.5%, respectively) localized in the wet season. The pregnancy rate between 57.1% and 61.4% suggests an estimated yearly production of 1.37–1.48 parturitions and a long estimated farrowing interval of 247–266 days. Although large antral follicles were observed in all females, pregnant females had a greater number of antral follicles than females in the luteal phase. The average litter size was 1.03 foetuses per pregnant female, and mean ovulation rate was 1.33 follicles, resulting in a rate of reproductive wastage of 28.7%. The constituent active luteal tissues of the ovary were oestrous cyclic, pregnancy and accessory CL. The 50% of pregnant females in the late pregnancy stage lacked pregnancy CL, suggesting that placenta may become the mean source of progesterone during late stages of pregnancy. Results of the present study suggest that the observation of the vaginal closure membrane should not be an accurate tool for diagnosing oestrus in the paca female.

Short communication: Change in dose delivery of prostaglandin F2α in a 5-day timed artificial insemination program in lactating dairy cows

We hypothesized that 50mg of prostaglandin F2α (PG) on d 6 would induce luteolysis in a traditional 5-d Ovsynch-72 program [GnRH 5 d before (d 0) and 72h after (d 8) 25-mg PG doses (d 5 and 6 after GnRH); timed artificial insemination (AI) on d 8]. Experiment 1 monitored luteal regression of original and GnRH-induced luteal tissue (corpus luteum, CL) by transrectal ultrasonography and blood serum concentrations of progesterone after both 25-mg doses of PG (d 5 and 6; control; n=31) or a single 50-mg dose of PG on d 6 (n=30). Estrous cycles were presynchronized (GnRH 7d before 25mg of PG); 11d later, cows were enrolled in a 5-d Ovsynch-72 program (62 to 71d in milk) and treatments were administered. Blood was sampled for progesterone analysis and luteal structures were measured on d 0 (original CL) and d 5 through 9 to monitor original and new GnRH-induced CL. Control PG reduced luteal tissue area of original CL on d 6 and 7 compared with PG administered only on d 6, but no difference between treatments was detected by d 9. In contrast, no differences were detected in luteal tissue area of the induced CL on d 5 through 9. Serum progesterone on d 5 through 9 differed only on d 6 for control and the 50-mg dose. Luteolysis occurred in all 31 controls, but luteolytic failure occurred in 2 of 30 cows receiving 50mg, in which no CL were present on d 0 but 1 or 3 new CL were present on d 5 in these 2 cows. Pregnancy outcome 32d after AI was 14 of 30 (40%) compared with 15 of 30 (50%) for control versus 50-mg dose, respectively. Experiment 2 monitored luteolysis in nonpregnant repeat-service cows subsequently treated with the same 2 treatments as in experiment 1. Serum progesterone in 63 cows (serum progesterone ≥1 ng/mL on d 5) on d 5, 6, and 8 differed only on d 6 for control and the 50-mg dose. Luteolysis occurred in 32 of 34 controls and in 29 of 29 cows treated with 50mg. Pregnancy outcome 32d after AI was 17 of 33 (52%) compared with 13 of 29 (45%) for control versus 50-mg dose, respectively. We concluded that the single 50-mg dose was equivalent to the control based on actual luteal tissue regression and decreased progesterone.