P4-treated Group Research Articles

Histomorphometric analysis of goat uterine tissue on in vitro exposure with ovarian hormones and mifepristone

Uterus, the largest reproductive tract organ in female mammals, is the site of implantation of fertilised egg and foetus development. Uterus is a dynamic reproductive organ; its morphology alters with reproductive phase and steroidal cues. The aim of the present study was to assess the effects of progesterone (P4), estrogen (E2) and antiprogestogen i.e., mifepristone on goat’s uterine histoarchitecture in in vitro short term culture. Uterine tissue slices were cultured in the presence of E2, P4 and mifepristone at the dose of 10–9 M, 10–7 M and 10–6 M respectively for 24 hours. Uter-ine morphology of E2- and P4-treated groups did not reveal marked changes from that of control group. Mifepristone treatment caused conspicuous changes in uterine histoarchitecture, led to congested endometrium, regressed uterine glands and constricted blood vessels. The changes ob-served in morphometry after E2 and P4 exposure included increased uterine gland diameter (47.00 and 45.95 µm respectively) and glandular epithelial cell height (18.37 and 17.43 µm respectively) while the mifepristone treatment resulted in significant reduction of gland diameter (34.95 µm) as well as epithelium height (14.25 µm) as compared to those in control group (39.9 and 15.56 µm respectively). These morphometrical changes revealed prominent regressive changes in anti-progestin treated group while E2 and P4 showed prolific effects in in vitro culture. Thus it is envis-aged that E2 and P4 induced characteristic progressive changes in the histologic structure especially in endometrial glands of the goat uterus while anti-steroidogenic formulation i.e. mifepristone severely reduced the normal histoarchitecture of the uterus which is a prerequisite for implanta-tion.

172 Expression of proliferation and apoptosis markers in cumulus cells surrounding matured and aged oocytes exposed to luteotropic factors during the second phase of in vitro maturation

The quality and developmental capacity of mammalian oocytes depends on cooperation with surrounding cumulus cells. The functional state and activity of cumulus cells changes with oocyte maturation, especially during the oocyte transition from metaphase I (MI) to metaphase II (MII) stage. In the present work, effects of 3 luteotropic factors, progesterone (P4), prolactin (PRL), and LH, during the second phase of in vitro maturation (IVM) on the subsequent expression of proliferation and apoptosis markers in bovine cumulus cells surrounding matured and aged oocytes were studied. A total of 1532 cumulus-oocyte complexes (COC) were cultured for 12h in TCM-199 containing 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, COC were transferred to the following IVM systems: (1) TCM-199 containing 10% FCS (Control 1) and (2) a monolayer of granulosa cells (GC) precultured for 12h in TCM-199 containing 10% FCS (Control 2). In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (50ng mL−1) or ovine LH (5μg mL−1); then, the COC were matured for next 12h. Half of the COC matured for 12h in both systems were cultured for an additional 24h in fresh TCM-199 containing 10% FCS to test long-term hormonal effects during oocyte aging. After culture, the cumulus expression of the proliferation marker proliferating cell nuclear antigen (PCNA) and the pro-apoptotic markers caspase-3 and Bax was assessed by the immunocytochemical method. The data from 4 to 5 replicates using 84 to 106 COC per treatment were analysed by ANOVA. After IVM in System 1, the rate of PCNA-positive cumulus cells was higher (P<0.05) in the PRL-treated group (41.3±1.6%) than in the control (34.6±2.3%) or LH-treated group (29.9±2.9%), but did not differ from that in the P4-treated group (38.2±4.8%). In the presence of GC (System 2), the respective rates did not change but were more variable. Aging of COC matured in both systems led to a 1.4- to 1.9-fold reduction in the proportion of the cells containing the proliferation marker PCNA (P<0.05). Meanwhile, none of the hormones tested had any long-term effect on the proliferative activity of senescent cumulus cells. The rate of cumulus cells expressing caspase-3 in different groups varied from 48.5±4.9 to 53.8±5.8% and did not depend on the hormones, IVM system, or oocyte aging. The proportion of the Bax-positive cells was also unaffected by luteotropic factors but increased 1.4 to 1.6 times (P<0.01) following 24h of COC aging. Our findings indicate that PRL can exert a short-term stimulatory action on the proliferative activity of bovine cumulus cells in the course of the second phase of IVM. Meanwhile, the cumulus expression of pro-apoptotic markers caspase-3 and Bax is not responsive to P4, PRL, or LH during the second step of IVM. The study was supported by the Russian Science Foundation (project 16-16-10069).

Interaction of the EGFR signaling pathway with porcine cumulus oocyte complexes and oviduct cells in a coculture system.

It has become increasingly recognized that coculture has a beneficial effect on the in vitro maturation (IVM) of oocytes and embryo development in many species. However, these effects of coculture on IVM have been documented only for their positive conditioning roles without any evidence on the precise mechanisms underlying the action of coculture systems on the development of cumulus oocyte complexes (COCs). It has been suggested that the epidermal growth factor receptor (EGFR) signaling pathway is important for development of COCs, mediated by several epidermal growth factor (EGF)-like proteins with downstream mitogen-activated protein kinase 1/3 signaling. Therefore, we hypothesized that canine oviduct cells (OCs) in a coculture system, which shows improvement of oocyte quality in several species, are associated with EGFR signaling by exposure to progesterone (P4; imitating its production before ovulation and its continuous increase while oocytes reside in the oviduct to complete maturation in dogs). We designed three experimental groups: control, OCs coculture exposed to P4, and OCs coculture without exposure to P4. The result showed that the OCs coculture exposed to P4 strongly expressed EGF-like proteins and significantly improved COCs and subsequent embryo development. Furthermore, the expression of EGFR-related genes in cumulus cells and GDF9 and BMP15 in oocytes was upregulated in the P4-treated group. This study provides the first evidence that OCs exposed to P4 can induce strong expression of EGF-like proteins, and OCs effectively mediate improved porcine COCs development and subsequent embryo development by altering EGFR signaling related mRNA expression.

Development of an in vitro model to study uterine functions and early implantation using rat uterine explants.

This study was conducted to develop an in vitro model using rat uterine explants to explore complex uterine functions. Rat uterine explants (1-2mm) were isolated, cultured and further characterized. Steroid hormone treatment of cultured explants showed that both Muc1 and Pr were significantly up-regulated (P<0.05) by E2. Areg was significantly up-regulated (P<0.05) by P4 and Igfbp1 was significantly up-regulated (P<0.05) by the combination of E2 and P4, although, in rat, Igfbp1 is E2-dependent. In vitro decidualization of cultured explants was induced and two potential markers of decidualization, Prl8a2 and Bmp2, were examined. Real-time quantitative PCR data revealed that both Prl8a2 and Bmp2 were significantly up-regulated (P<0.05) in MPA- and db-cAMP-treated explants compared to the control group of explants. Then, an individual hatched blastocyst and cultured explant was placed in a 96-well(round-bottom U-shaped) plate. Co-culture results showed that stable attachments were observed after 48h, where embryos were stably attached to the explants and could not be dislodged after mild shaking and/or pipetting. The rates of attachment of embryos to the explants were increased significantly in the P4-treated group (63.6%) compared to the control group (35.5%), after steroid hormone treatment. The rates of attachment were reduced significantly in the E2-treated group (0.0%), where no stable attachments were observed. Despite the necessity of comprehensive investigation, our results suggest that the cultured rat uterine explants can be a useful in vitro model to study uterine functions and early implantation.

Progesterone modulates diabetes/hyperglycemia-induced changes in the central nervous system and sciatic nerve

We investigated the effect of progesterone (P4) treatment on diabetes/hyperglycemia-induced pathological changes in brain, spinal cord and sciatic nerve tissue in male rats. Animals were rendered hyperglycemic by a single dose of streptozotocin (STZ). P4 treatment was started after hyperglycemia was confirmed and body weight and blood glucose levels were monitored once/week for 5weeks. Rats underwent behavioral testing at week 5 and were then euthanized for histology. We assessed the expression of markers of angiogenesis (vascular endothelial growth factor (VEGF)), inflammation (interleukin-6 (IL-6)) and tissue injury (CD11b, NG2, COX2 and matrix metalloproteinase-2 (MMP-2)) in the brain, spinal cord and sciatic nerve. We also examined the regenerative effect of P4 on pathological changes in intra-epidermal nerve fibers (IENF) of the footpads. Diabetes/hyperglycemia led to body weight loss over 5weeks and P4 treatment reduced this loss. At week 5, blood-glucose levels were significantly lower in the P4-treated diabetic group compared to vehicle. Compared to sham or P4-treated groups, the diabetic vehicle group showed hyperactivity on the spontaneous locomotor activity test. Western blot data revealed upregulation of VEGF, IL-6, CD11b, NG2, COX2 and MMP-2 levels in the vehicle group and P4 treatment normalized these expression levels. IENF densities were reduced in the vehicle group and normalized after P4 treatment. We conclude that P4 can reduce some of the chronic pathological responses to STZ-induced diabetes.

Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro.

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

The effect of steroid hormones on the mRNA expression of oct4 and sox2 in uterine tissue of the ovariectomized mice model of menopause

The uterus is a dynamic tissue responding to hormonal changes during reproductive cycles. As such, uterine stem cells have been studied in recent years. Transcription factors oct4 and sox2 are critical for effective maintenance of pluripotent cell identity. The present research evaluated the mRNA expression of oct4 and sox2 in the uterine tissues of ovariectomized mice treated with steroid hormones. In this experimental study, adult virgin female mice were ovariectomized and treated with estradiol 17β (E2), progesterone (P4), and a combination of E2 and P4 (E2 & P4) for 5 days. Uterine tissues were removed, and immunofluorescent (IF) staining and quantitative real-time PCR of oct4 and sox2 markers were performed. IF showed oct4 and sox2 expression in the uterine endometrium and myometrium among all groups. The mRNA expression of oct4 (p=0.022) and sox2 (p=0.042) in the E2-treated group significantly were decreased compared to that in the control group. By contrast, the mRNA expression of oct4 and sox2 in the P4 (p=0.641 and 0.489 respectively) and E2 & P4-treated groups (p=0.267 and 0.264 respectively) did not show significant differences compared to the control group. The results indicate ovarian steroid hormones change the expression of oct4 and sox2 in the mice uterine tissues, which suggest the involvement of steroid hormonal regulation in uterine stem cells.

11 FERTILITY RESPONSE IN SUCKLED BEEF COWS SUPPLEMENTED WITH LONG-ACTING PROGESTERONE AFTER TIMED ARTIFICIAL INSEMINATION

Corpus luteum (CL) and progesterone (P4) secretion are affected by preovulatory follicle (POF) size. Increased circulating P4 during early diestrus has a positive effect on embryo development in beef cattle. However, the combined effects of the POF size and P4 supplementation during early diestrus on fertility of beef cows are not known. The objective was to evaluate the effects of POF size and supplementation of long-acting P4 after timed-AI on pregnancy rates (P/AI). Suckled Nelore cows (n = 596) were evaluated twice by transrectal Doppler ultrasonography (US) 10 days apart to detect the cyclic status. In Study 1, anestrous cows (absence of CL on both exams) received an intravaginal P4 device and an oestradiol benzoate (EB) injection on Day –10 (day of the second US). Devices were removed and sodium cloprostenol [prostaglandin F2α (PGF2α)], oestradiol cypionate, and eCG were given on Day –2. Cows were timed-AI on Day 0 and assigned to receive placebo (control group, n = 187) or 150 mg of long-acting P4 on Day 4 (P4 group, n = 189). In Study 2, cyclic cows (presence of CL) received a PGF2α injection on Day –20 (first US). Cows with a new CL on Day –10 received an intravaginal P4 device and an injection of EB and were split to receive an injection of PGF2α [large follicle (LF); n = 109] or not [small follicle (SF); n = 111]. Devices were removed and PGF2α was injected on Day –2. Ovulation was induced with buserelin acetate, and cows were timed-AI on Day 0 and split to receive placebo (LF/control group, n = 55, and SF/control group, n = 55) or 150 mg of long-acting P4 on Day 4 (LF/P4 group, n = 56, and SF/P4 group, n = 54). Ultrasonographic scanning was done on Days 0, 4, and between 35 and 40 to detect the POF and CL sizes and P/AI, respectively. Data were analysed using PROC GLIMMIX (SAS Institute Inc., Cary, NC). In anestrous cows, P/AI was reduced in POF with &lt;11 mm. The P/AI was greater in the P4-treated group than in the control group for all cows (55.6% v. 46.0%; P = 0.05) and for ovulated cows (59%, 105/178 v. 49%, 86/173; P = 0.08). For cyclic cows, POF size (mm) on Day 0 (13.5 ± 0.3 v. 11.2 ± 0.2), ovulation rate (90% v. 77%), and CL area (cm2) on Day 4 (1.46 ± 0.05 v. 1.25 ± 0.05) were greater (P &lt; 0.007) in the LF group than in the SF group. There was a main effect of follicle group on P/AI (54%, LF group v. 38%, SF group; P &lt; 0.01). Moreover, P/AI were greater (P &lt; 0.05) in the LF/control (56%) and LF/P4 (52%) groups than in the SF/control group (31%), whereas no difference was detected between the SF/P4 group (45%) and the other groups. Among cows that ovulated, P/AI was lower (P = 0.05) in the SF/control group (41%, 17/41) compared with the LF/control group (62%, 31/50) and were similar for the SF/P4 group (56%, 25/45) and LF/P4 group (57%, 28/49) compared with others. We suggest that P4-stimulated embryotrophic effects improved fertility in anestrous beef cows supplemented with long-acting P4 on Day 4 after timed-AI. Also, the presence of a functional CL during follicle growth results in smaller POF and CL and reduces the ovulatory and P/AI rates in cyclic cows. Post-AI P4 supplementation may attenuate the negative effects of small POF/CL. Research was supported by CNPq, FAPESP, Ouro Fino Agronegócio, and Innovare.

Corpus Luteum Development and Function after Supplementation of Long‐Acting Progesterone During the Early Luteal Phase in Beef Cattle

Strategic supplementation of P4 may be used to increase conception rates in cattle, but timing of supplementation in relation to ovulation, mass of supplementary P4 and formulation of the P4-containing supplement has not been determined for beef cattle. Effects of supplementation of long-acting progesterone (P4) on Days 2 or 3 post-ovulation on development, function and regression of corpus luteum (CL) were studied in beef cattle. Cows were synchronized with an oestradiol/P4-based protocol and treated with 150 or 300mg of long-acting P4 on Day 2 or 3 post-ovulation (6-7 cows/group). Colour-doppler ultrasound scanning and blood sample collection were performed from Day 2-21.5. Plasma P4 concentrations were greater (p<0.05) from Day 2.5-5.5 in the Day 2-treated groups and from Day 3.5-5.5 in the Day 3-treated cows than in the control group. CL area and blood flow during Day 2-8.5 did not differ (p>0.05) among groups, suggesting no effect of P4 treatment on luteal development. The frequency of cows that began luteolysis before Day 15 was greater (p<0.04) in cows treated with 300mg than in the controls, but there were no differences between non-treated and 150mg-treated cows. The interval from pre-treatment ovulation to functional and structural luteolysis was shorter (p<0.01) in the combined P4-treated groups than in the control cows. In conclusion, was showed for the first time that long-acting P4 supplementation on Day 2 or 3 post-ovulation increases P4 concentrations for ≥3day, has no effect on luteal development, but anticipates the beginning of luteolysis in beef cattle.

Actions of oestradiol and progesterone on the prostate in female gerbils: reversal of the histological effects of castration

The female prostate is a functionally active gland in several mammalian species, including humans and rodents. Investigations of prostate morphophysiology during the phases of the oestrous cycle have shown that the female prostate is influenced by fluctuations in serum concentrations of oestradiol (E2) and progesterone (P4). The aim of the present study was to evaluate the effect of combined prolonged administration of E2 and P4 on the prostate in ovariectomised female gerbils. Ovariectomy caused atrophy and decreased glandular secretory activity. Administration of E2 and P4 (0.1mgkg(-1) diluted in 0.1mL of mineral oil, every 48h over 30 days) resulted in a recovery of overall prostate structure, as evidenced by increased epithelial height, mass and prostatic secretory activity, without leading the appearance of significant lesions. Evaluation of androgen receptor (AR) expression revealed increased immunoreactivity in the E2+P4-treated group. Immunostaining for oestrogen receptor (ER) α was decreased in the castrated groups, but increased in the group subjected to hormone treatment. There were no significant differences in ERβ immunoreactivity among the groups. Assessment of cell proliferation revealed greater immunoreactivity in the treated group. Together, the results indicate that the interaction between E2 and P4 may be responsible for maintaining female prostate gland histophysiology.

Abstract TMP97: Progesterone Attenuates Acute Seizure and Brain Infarction by Modulating Pro-Inflammatory Cytokines in a Mouse Model of Neonatal Stroke

Neonatal stroke is among the top ten causes of childhood death and causes long-term permanent disability and neurological deficits in survivors. In most cases, recurrent clonic seizure is the only clinical manifestation which worsens the stroke outcomes. A treatment or pediatric stroke is essential, but no candidate intervention is supported by substantiated data. We tested the hypothesis that progesterone (P4) treatment would be beneficial in a neonatal model of stroke. P12 CD1 mice (mixed gender) underwent permanent unilateral right common carotid ligation (pUCCL) or sham surgery (n=10/group). Pups which showed seizure activity during the 1h post-pUCCL were randomly assigned to receive P4 (8 mg/kg) or vehicle injections at 1, 3 and every 24h post-pUCCL for 6 days. We assessed acute behavioral seizures (during the first 4h post-pUCCL), serum pro-inflammatory cytokines (IL-1β, IL-6, TNFα at 6, 24 and 48h) and brain infarction (at day 7 by CV-staining). Cytokine data were analyzed by repeated measures one-way ANOVA followed by LSD and Tukey’s tests for independent comparisons. For seizure and infarct data, a two-tailed unpaired t-test was employed. We observed acute seizures during the first 4h post-pUCCL in the vehicle group (90.8±11.77). P4 treatment significantly ( P &lt;0.05) reduced seizure occurrence (58.4±8.97) by 35% compared to vehicle. Repeated measures ANOVA revealed a significant group effect in IL-1β (F (2,15) =110.706; P &lt;0.001), IL-6 (F (2,15 ) =66.067; P &lt;0.001), and TNFα (F (2,15) =146.263; P &lt;0.001) levels. Serum IL-1β, IL-6 and TNFα were significantly higher ( P &lt;0.001) at 6 and 24h and remained elevated until 48h post-pUCCL in the vehicle group compared to sham. The P4-treated group showed a significant ( P &lt;0.01) decrease in all pro-inflammatory cytokine levels at all time points. Further, pUCCL resulted in severe hemispheric damage (16.88±1.48) as evidenced by cresyl-violet staining at 7 days post-pUCCL. P4 treatment showed a significant ( P &lt;0.05) reduction (4.93±1.1) in infarct volume (~70%) compared to vehicle. Our data demonstrate that P4 reduces acute seizures and brain infarction following neonatal stroke by modulating the inflammatory process, and warrants detailed studies of functional outcomes and mechanism of action.

Endogenous and exogenous progesterone influence body temperature in dairy cows

Three experiments were conducted to determine the effect of endogenous progesterone (P4) on body temperature comparing lactating, pregnant with lactating, nonpregnant cows, and to study the effect of exogenous P4 administered via a controlled internal drug release (CIDR) insert on body temperature in lactating dairy cows. Body temperature was measured vaginally and rectally using temperature loggers and a digital thermometer, respectively. In experiment 1, 10 cyclic lactating cows (3 primiparous, 7 multiparous) and 10 lactating, pregnant cows (3 primiparous, 7 multiparous) were included. Vaginal temperatures and serum P4 concentrations were greater in pregnant cows (vaginal: 0.3±0.01°C; P4: 5.5±0.4 ng/mL) compared with nonpregnant cows. In experiment 2, estrous cycles of 14 postpartum healthy, cyclic, lactating cows (10 primiparous, 4 multiparous) were synchronized, and cows were assigned randomly to 1 of 2 treatments (CIDR-P4 or CIDR-blank). A temperature logger was inserted 1 d after ovulation using a P4-free CIDR (CIDR-blank) and a CIDR containing 1.38g of P4 (CIDR-P4) in the control (n=7) and the P4-treated group (n=7), respectively. On d 3 after P4 treatment, vaginal temperature was 0.3±0.03°C greater compared with that on d 1 and d 5. In experiment 3, 9 cyclic multiparous lactating cows were enrolled 1±1 d after confirmed ovulation and a temperature logger inserted. Two days later, a CIDR-P4 was inserted on top of the CIDR-blank. On d 5±1 and d 7±1, respectively, the CIDR-P4 and CIDR-blank with the temperature logger were removed. During the CIDR-P4 treatment (48h), vaginal temperature was 0.2±0.05°C and 0.1±0.05°C greater than during the pre- and post-treatment periods (48h), respectively. Serum P4 concentration peaked during CIDR-P4 treatment (2.2±0.8 ng/mL) and was greater than during the pre-treatment period (0.2±0.2 ng/mL) for 48h. An increase in vaginal temperature could be due to endogenous and exogenous P4. However, a correlation between serum P4 concentrations and body temperature did not exist. Further investigations are warranted to better understand the pathways of the thermogenic effect of P4 on body temperature.

Progesterone-Treated Mares Ovulate in Presence of Low Systemic Concentrations of LH.

Mares are useful comparative research models for studying ovarian dynamics in women because of similarities in the number and nature of follicular waves and a constant relative diameter of the largest follicle between the two species at definable events throughout the ovulatory wave. Recent (2005 to 2009) temporal and mechanistic studies have contributed to the understanding of the hormonal changes during the periovulatory period in mares. However, regulatory mechanisms of ovulation in mares are not well known, especially, the role of the long secretory pattern of luteinizing hormone (LH) during the periovulatory period. Progesterone (P4) treatment for several days during the estrous cycle may depress gonadotropins concentrations, block follicular development, and induce persistent anovulatory follicles in several species. The goals of this study were: (1) to test if a high dose of exogenous progesterone (P4) given during the preovulatory period reduces the LH surge concentrations and blocks ovulation in mares with single or double dominant follicles; and (2) to study the effect of P4 treatment on follicle blood flow. Mares with a growing ≥25-mm follicle 15 days after ovulation were scanned daily by ultrasonography. When the largest follicle reached ≥30 mm and the endometrial echotexture score was indicative of preovulatory period, mares were randomly assigned to one of the following four groups (n=8-10 mares/group): single dominant control (SD); single dominant+progesterone (SD+P4); double dominant control (DD); and double dominant+progesterone (DD+P4). The treatments involved the vehicle (3 ml of safflower oil) for the control groups and P4 (150 mg in 3 ml of safflower oil) for the P4-treated groups. Treatment was given every 12 h for 7 days. Ovulation of the largest follicle occurred in 100, 38, 100, and 56% of the mares in the groups SD, SD+P4, DD, and DD+P4, respectively. Ovulation of the second largest follicle occurred in 44 and 11% in the groups DD and DD+P4. Follicle atresia or formation of a hemorrhagic anovulatory follicle was the outcome for follicles that did not ovulate. Plasma LH concentrations but not FSH were significantly (P<0.0001) inhibited by P4 treatment. Mares in the P4-treated groups ovulated in presence of very low (7-fold less) concentrations of LH when compared with the controls. Diameter of the largest ovulatory follicle and estradiol concentration did not differ among groups. Blood flow in the wall of the largest follicle was less (P<0.04) in P4-treated groups 3 days after beginning of treatment. In conclusion, the preovulatory LH surge does not seem to be crucial for ovulation to occur in mares. From a clinical standpoint, attention should be given to progesterone-based synchronization or protocols for inhibition of estrous behavior since progesterone-treated mares may ovulate with very low systemic concentrations of LH. The present findings may also have implications in human reproductive medicine due to the striking similarities between mares and women in several aspects of folliculogenesis. (poster)

16 MODIFICATION TO INTRAVAGINAL PROGESTERONE + ESTRADIOL-BASED ESTROUS SYNCHRONIZATION PROTOCOLS TO IMPROVE OVARIAN RESPONSES IN BOS INDICUS HEIFERS

Estrous synchronization (ES) protocols for fixed-time AI (FTAI) have been mainly developed for Bos taurus cattle assuming that usage in Bos indicus cattle results in similar outcomes. Unexplained variation in the reproductive outcomes of these treatments in extensively managed Bos indicus herds has limited the adoption of FTAI. The aim was to evaluate the ovarian function in heifers treated with different doses of progesterone (P4). Brahman (n = 30) and Brahman-cross (n = 34; 3/4 to 7/8 Brahman) heifers weighing an average of 285 kg (248 to 323 kg) and 307 kg (250 to 363 kg), respectively, were allocated at random to 1 of 4 treatment groups: 1) full-dose Cue-Mate (CM100; two P4-impregnated pods, 1.56 g of P4; Bioniche Animal Health Aust/Asia, Armidale, NSW, Australia; n = 17); 2) half-dose Cue-Mate (CM50; one P4-impregnated pod; 0.78 g of P4; n = 15); 3) half-dose Cue-Mate + 300IU of eCG (CM50+G; Pregnecol, Bioniche Animal Health, Aust/Asia; n = 14) i.m. at Cue-Mate removal; and 4) double PGF2α group (PG; n = 18). Heifers in the P4-treated groups received 1 mg of EB (estradiol benzoate; Ciderol; 1 mg mL-1; Genetics Australia, Bacchus Marsh, Victoria) at Cue-Mate insertion (Day -10) and 24 h after Cue-Mate removal (Day -1) and 250 μg of PGF2α (Cloprostenol, Estromil, Ilium Veterinary Products, Smithfield, Australia) i.m. at Cue-Mate insertion (Day -10) and removal (Day -2). Heifers in the PG group received 500 μg of PGF2α on Day -16 and Day -2 i.m. Ovarian function was evaluated twice daily by transrectal ultrasonography on Day -1, Day 0 (expected estrus), and Day 1, and once daily on Days 4, 6, 8, 10, 12, and 16 of the synchronized and return cycles. Concurrent blood samples were collected to determine plasma P4 concentrations. A one-way ANOVA was used to analyze follicle diameter variables and Fisher’s Exact test was used to analyze all other data reported (StataCorp 2007, College Station, TX, USA). There was no difference (P = 0.31) in the diameter of the dominant follicle 54 to 56 h post-Cue-Mate removal (the time FTAI would be conducted) in the CM100 (11.5 ± 1.1), CM50 (13.3 ± 1.5), CM50+G (13.7 ± 1.5), or PG (14.5 ± 0.7) groups. Synchrony of ovulation in ovulating heifers did not differ (P &lt; 0.05) between the P4-treated groups with 12/13 (CM100), 11/12 (CM50), and 9/12 (CM50+G) heifers ovulating within 53 to 77 h after Cue-Mate removal, but was lower (P &lt; 0.02) for the PG group (1/5). Individual heifer P4 profiles revealed that 2/15, 1/15, 3/12, and 0/5 of heifers that ovulated in the CM100, CM50, CM50+G, andPG groups, respectively, failed to re-ovulate in the return cycle. Also, 1/15, 3/15, 1/12, and 0/5 of heifers that ovulated in the CM100, CM50, CM50+G, and PG groups, respectively, developed a CL that persisted into the return cycle. This study shows the potential benefits of using intravaginal devices containing a lower amount of P4 to induce synchronized ovulation in Brahman heifers, consistent with the findings of Carvalho et al. (2008 Theriogenology 69, 167).

Protection against Genital Herpes Infection in Mice Immunized under Different Hormonal Conditions Correlates with Induction of Vagina-Associated Lymphoid Tissue

The present study was undertaken to examine the effect of the hormonal environment on immunization with an attenuated strain of herpes simplex virus type 2 (HSV-2 TK(-)) and subsequent protection against challenge. Ovariectomized mice were administered saline (S; control), estradiol (E(2)), progesterone (P(4)), or a combination of estradiol and progesterone (E+P) and immunized intravaginally (IVAG) with HSV-2 TK(-). Three weeks later, the immunized mice were challenged IVAG with wild-type HSV-2. Mice that were immunized following E treatment were not protected, whereas complete protection against the challenge was seen in mice from the S- and P(4)-treated groups. In the P(4)-treated group, 15% of mice developed chronic pathology following TK(-) immunization. Interestingly, about 40% of the E+P-treated mice were also protected. Upon examination of viral shedding in the vaginal secretions, it was clear that protection against challenge was dependent on the ability of the TK(-) virus to cause productive genital infection under different hormonal conditions. In the protected mice (the S and P groups and part of the E+P group), induced vagina-associated lymphoid tissues composed of CD11c(+) dendritic cells and CD3(+) and CD4(+) T cells were formed transiently in the vaginal lamina propria from day 2 to day 5 postchallenge. These aggregates were absent in the unprotected mice (the E group and part of the E+P group). Significant HSV-2-specific activation of lymphocytes was observed in the local draining lymph nodes of protected mice. This response was absent in the unprotected groups. High titers of gB-specific local immunoglobulin A (IgA) antibodies were present in the vaginal secretions of S- and P(4)-treated immunized mice following HSV-2 challenge. The S-treated group of mice also had high gB-specific IgG titers. These studies show that sex hormones modify the induction of protective immune responses following IVAG immunization.

Estradiol Regulates Susceptibility following Primary Exposure to Genital Herpes Simplex Virus Type 2, while Progesterone Induces Inflammation

We report here that sex hormones modulate susceptibility to a sexually transmitted viral agent, herpes simplex virus type 2 (HSV-2), in a mouse model. Ovariectomized mice were administered either saline (control), estradiol (E(2)), progesterone (P(4)), or a combination of both estradiol and progesterone (E+P) and infected intravaginally with HSV-2. With an inoculation dose of 10(5) PFU, the saline- and P(4)-treated mice were found to be highly susceptible to genital HSV-2 infection. Both groups had extensive pathology and high viral titers in vaginal secretions, and 100% of mice succumbed by day 4 postinfection. E(2)-treated mice were protected from HSV-2 infection at the same dose and did not display any vaginal pathology or viral shedding. There was a slow progression of genital pathology in the combination hormone-treated group, along with prolonged viral shedding; 80% of animals succumbed by day 13. With lower inoculation doses of 10(3) and 10(2) PFU, 50 and 100%, respectively, of the combination hormone-treated mice survived. Localization of HSV-2 infection showed extensive infection in the vaginal epithelium of P(4)- and saline-treated animals within 24 h of inoculation. E(2)-treated animals were clear of infection, while the E+P-treated group had focal infection at 24 h that had progressed extensively by day 3. Infection was accompanied by persistent inflammation and infiltration of neutrophils in the P(4)-treated group. An analysis of the genes in the vaginal tissue showed that inflammation in the P(4)-treated group correlated with local induction of chemokines and chemokine receptors that were absent in the E(2)-treated mice and in uninfected P(4)-treated mice. The results show that sex hormones regulate initiation of infection and immune responses to genital HSV-2 infection.

Progesterone-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index.

The objective was to determine whether there were decreases in insulin-like growth factors (IGF)-I and -II and increases in low-molecular-mass IGF-binding proteins (IGFBPs) in association with an inhibition of aromatase activity (AA) and follicular fluid estradiol (E2) production during progesterone (P4)-induced dominant follicle atresia in cattle. Twelve cycling cows received a norgestomet ear implant at proestrus for 9 days and were assigned to control (n = 3) or P4-treated (n = 9) groups. Injections of P4 (150 mg, i.m.) were given on Days 3 and 4; Days 3, 4, and 5; or Days 3, 4, 5, and 6 of the implant period. Controls received injections of corn oil on Days 3, 4, 5, and 6. Ultrasonography of the dominant follicle and blood sampling were done daily. Unilateral ovariectomy was done one day after the last injection. The experiment was repeated with the remaining ovary (6 follicles/treatment group). Granulosa cells were cultured with radiolabeled testosterone to measure AA. Steroid hormones, IGF-I, and IGF-II were measured in follicular fluid by RIA. The follicular fluid IGFBP profile was quantified by Western ligand blotting. P4-treated cows showed a drastic reduction in AA in the dominant follicles, and follicular fluid E2 was several times lower than in controls. Moreover, in P4-treated groups, concentrations of follicular fluid IGF-I and IGF-II were lower than in controls. The quantity of low-molecular-mass follicular fluid IGFBPs increased in P4-treated groups. Accumulation of low-molecular-mass IGFBPs with a reduction in IGFs may be a mechanism of dominant follicle atresia during the bovine estrous cycle.

Steroid hormone effects on intercellular communication between term pregnant human myometrial cells before labor.

The appearance of gap junctions (GJs) between myometrial smooth muscle cells is one of the major events associated with the onset of labor. We have employed dye-coupling and electrical-current injection techniques to study the mechanisms by which steroid hormones regulate GJs in term pregnant myometrium of women before labor. Progesterone (P4) did not alter the input resistance (Ro) of the tissues when added to Tyrode's solution, which was used as control treatment. Octanol, the putative gap junctional uncoupling agent, increased the Ro of the cells compared to the control and P4-treated groups. The membrane potential (Em) did not differ between these groups. However, when P4 was applied after the tissue was perfused with estradiol (E2), the results changed dramatically: the Em hyperpolarized, and the Ro increased. Octanol increased the Ro in E2-treated tissues, but did not affect the Em. Consecutive application of E2, octanol, E2, and P4 resulted in rapid changes in the Ro of the cells. Dye-coupling was mostly detected between cells from controls and E2-treated tissues. These results indicate that P4 exerts its effects in the presence of E2 and that P4 has rapid effects on the intercellular communication between human myometrial cells.

Cellular localization and sex steroid regulation of insulin-like growth factor binding protein messenger ribonucleic acids in the primate myometrium.

Insulin-like growth factors (IGF)-I and -II are abundant in the primate myometrium and are implicated in the sex steroid-induced growth of this tissue. To evaluate the potential for modulation of IGF action in the primate myometrium by locally produced IGF binding proteins (IGFBPs), we examined the cellular localization and sex steroid regulation of IGFBPs 1-5 in the nonhuman primate uterus. Ovariectomized rhesus monkeys were treated with placebo, estradiol (E2), or E2 and progesterone (P4) (E2 and P4) for 2 weeks, after which their uteri were removed and cut into thin serial sections for analysis by in situ hybridization. IGFBP-1 messenger RNA (mRNA) was not detected in control or E2-treated uteri but was found in a few unidentified cells in the E2 and P4-treated group. IGFBP-2, -3, -4, and -5 mRNAs were all expressed by myometrial smooth muscle cells but each displayed distinctive patterns of regulation by sex steroids. IGFBP-2 was barely detectable in control myometrium, was significantly increased by E2 and even more significantly by E2 and P4. IGFBP-4 and 5 mRNAs were readily detectable in control myometrium and significantly increased by E2 treatment. The addition of P4 to E2 treatment did not produce a significantly greater augmentation in IGFBP-4 or 5mRNA level compared with E2 alone. IGFBP-3 mRNA was abundant in the control myometrium, but in contrast to other IGFBPs was significantly reduced by approximately 75% in smooth muscle cells by E2 and by E2 and P4 treatment. Interestingly, however, IGFBP-3 mRNA was increased in the uterine vascular endothelium by E2 and by E2 and P4 treatment. In summary, this study has shown that four of the six known IGFBPs are highly expressed in the primate myometrium, and that their expression is differentially regulated by sex steroids. The cellular and sex steroid-regulated pattern of IGFBP-2 gene expression is very similar to that of IGF-I, as previously determined in these same myometrial samples. Both IGF-I and IGFBP-2 are dependent on E2 for significant myometrial expression, and both are further augmented by the addition of P4 to E2 treatment. Uterine smooth muscle IGFBP-3 mRNA levels are negatively regulated, whereas IGFBP-4 and -5 mRNA levels are positively regulated by E2: none of these myometrial IGFBPs appears sensitive to the effects of P4. The present observations, together with our previous data from the same animals, demonstrate that the primate myometrial smooth muscle cell expresses mRNAs for IGF-I and -II, IGF-I and -II receptors, as well as expresses mRNAs for IGFBP-2, -3, -4, and -5. These data provide evidence for complex local interactions between IGF system components regulated by estrogen and progesterone.

The postpartum induced corpus luteum: functional differences from that of cycling cows and the effects of progesterone pretreatment.

Anestrous postpartum (PP) Hereford cows (n = 41) were used to compare corpora lutea (CL) from gonadotropin-releasing hormone (GnRH)-induced ovulation with CL from cycling cows. Postpartum cows were injected i.m. daily with 100 mg progesterone (P4) or oil on Days 25 through 28 PP and then given 200 micrograms GnRH i.m. on Day 30 PP. Corpora lutea were removed from one-half of the PP cows in the oil- and P4-treated groups 6.5 days after GnRH injection, and from the cycling cows 7 days after estrus. Intact PP cows were used to evaluate cycle length. Blood was collected daily from all PP cows from Day 25 PP through luteectomy and on Days 9, 11, and 13 post-GnRH from the oil- and P4-intact cows to determine short (SHORT) versus normal (NORM) luteal phases. Cycling cows were bled daily from estrus until CL removal NORM PP cows had higher (P less than 0.001) P4 levels than did SHORT PP cows from Day 7 through Day 13 post-GnRH, and more (P less than 0.05) P4-intact cows were NORM compared with oil-intact cows (45.5% vs. 14.3%, respectively). Corpora lutea from cycling cows were heavier (P less than 0.05) and had a higher luteinizing hormone (LH) receptor concentration (P less than 0.05), but CL P4 concentration did not differ from PP cows. Corpora lutea weight, LH receptor and P4 concentration, and in vitro P4 production were similar in the oil-and P4-treated PP cows. NORM cows had heavier CL (P less than 0.05) than SHORT cows, although P4 content and LH receptor concentration did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)