ABSTRACT
The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.
Subject(s)
Estradiol/pharmacology , Horses/physiology , Luteinizing Hormone/biosynthesis , RNA, Messenger/metabolism , Animals , Blotting, Northern/veterinary , Estradiol/administration & dosage , Female , Luteinizing Hormone/blood , Luteinizing Hormone/genetics , Ovariectomy/veterinary , Pituitary Gland/drug effects , Pituitary Gland/metabolism , RNA, Messenger/genetics , Random Allocation , SeasonsABSTRACT
The potential involvement of ovarian factors in regulating GnRH and LH postovulation was studied in ovarian intact (Group 1; n=3) and ovariectomized (OVX; Group 2; n=3) mares (OVX within 12 hr of ovulation). Blood samples were collected every 10 min for 6 hr from jugular vein (JV) and intercavernous sinus (ICS) during estrus and on Day 8 postovulation for LH and GnRH analysis. Additionally, JV samples were collected twice daily (12-hr intervals) for 30 days for LH and progesterone (P4) analysis. A significant treatment x day effect (P<0.0001) describes declining plasma LH concentrations in intact mares, and regression analysis indicated that response curves were not parallel (P<0.001). Plasma LH concentrations remained elevated in OVX mares. LH increased further in OVX mares by Day 8 post-OVX (P<0.06), reflecting the increased (P<0.07) LH episode amplitude. GnRH decreased from estrus to Day 8 in both groups reflecting an effect of sampling period (P<0.03). GnRH episode amplitude declined (P<0.08) from estrus (62.8+/-3.1 pg/mL) to Day 8 (46.3+/-3.1 pg/mL) in OVX mares, but not in control mares (intact estrus, 36.5+/-6.4; intact Day 8, 37.5+/-7.3; OVX estrus, 62.8+/-3.1; OVX Day 8, 46.3+/-3.1 pg/mL). In conclusion, we propose that postovulatory LH decline requires ovarian feedback in mares, and that OVX alters GnRH secretory dynamics such that LH concentrations does not decline postovulation and, in fact, is further elevated with time after OVX.
Subject(s)
Gonadotropin-Releasing Hormone/metabolism , Horses/physiology , Luteinizing Hormone/metabolism , Ovary/physiology , Ovulation/physiology , Animals , Feedback , Female , Gonadotropin-Releasing Hormone/blood , Least-Squares Analysis , Luteinizing Hormone/blood , Ovariectomy/veterinary , Progesterone/blood , Random AllocationABSTRACT
Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 micrograms/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (D0) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 +/- 3.68 ng/ml: LSMEANS = SEM), than either Group 1 (2.65 +/- 4.64 ng/ml) or Group 3 (4.28 +/- 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 +/- 1.53 ng/ml) than Group 1 or Group 3 (8.34 +/- 1.84 and 7.69 +/- 1.63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery. 2) pulsatile administration of GnRH (25 micrograms/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.
Subject(s)
Estradiol/pharmacology , Follicle Stimulating Hormone/blood , Gonadotropin-Releasing Hormone/pharmacology , Horses/physiology , Luteinizing Hormone/blood , Pituitary Gland/surgery , Animals , Estradiol/administration & dosage , Female , Gonadotropin-Releasing Hormone/administration & dosage , Kinetics , Ovariectomy , Periodicity , Pituitary Gland/physiologyABSTRACT
This study compared equine and ovine LH secretory responses to GnRH treatment. Dioestrous mares and ewes were challenged with continuous GnRH for 15 h. Mares that received constant GnRH (110 micrograms h-1) had sustained LH secretion (P < 0.01), whereas LH concentrations in ewes treated with continuous GnRH (25 micrograms h-1) initially increased, then declined and remained low, suggesting GnRH receptor desensitization or downregulation. In addition, progesterone-primed, ovariectomized mares and ewes were challenged with pulsatile or continuous GnRH for 5 days. Plasma LH concentrations were increased by day 5 in mares treated with pulsatile (25 micrograms pulse-1 h-1) and continuous (110 micrograms h-1) GnRH (P < 0.01). Furthermore, mean LH concentrations and time-response curves were not different. In contrast, ewes treated with continuous GnRH (2.5 micrograms h-1) demonstrated LH secretory patterns indicative of GnRH receptor downregulation on day 1 of treatment. LH concentrations in ewes treated with pulsatile GnRH (250 ng pulse-1 h-1) did not differ from controls. In conclusion, pony mares responded continuously to GnRH treatment (pulsatile and continuous), whereas ewes treated with continuous GnRH experienced reduced LH secretion. These findings suggest a unique hypothalamic-pituitary axis in pony mares.
Subject(s)
Gonadotropin-Releasing Hormone/pharmacology , Horses/physiology , Luteinizing Hormone/metabolism , Sheep/physiology , Animals , Diestrus , Dose-Response Relationship, Drug , Down-Regulation , Female , Infusions, Intravenous , Luteinizing Hormone/blood , Ovariectomy , Progesterone/pharmacology , Random Allocation , Receptors, LHRH/metabolismABSTRACT
The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of "fixation," or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These "defective" conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with 3H on sialyl-oligosaccharides and 125I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule.
Subject(s)
Abortion, Veterinary , Extraembryonic Membranes/chemistry , N-Acetylneuraminic Acid/analysis , Prostaglandins/pharmacology , Abortion, Induced/veterinary , Animals , Autoradiography , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Female , Horses , PregnancyABSTRACT
A family of estrogen-dependent porcine oviductal secretory glycoproteins (POSPs) that exhibit structural similarities are synthesized and secreted into the oviductal lumen at proestrus, estrus, and metestrus. The objectives of this study were to clone the POSP cDNA, obtain the full-length cDNA and protein sequence, examine tissue specificity and species distribution, characterize its regulation, and establish its identity by comparison to other known protein, RNA, or DNA sequences. A full-length cDNA of 2022 base pairs was obtained with an open reading frame of 1581 nucleotides, coding for a deduced protein of 527 amino acids (57 970 M(r)). The deduced protein contained three potential N-glycosylation sites, a consensus heparin-binding site, and potential O-glycosylation sites. Amino acid analysis of POSP-E3 confirmed the presence of a 21-amino acid signal sequence. Northern blot analysis revealed an oviduct-specific mRNA species of 2.25 kb in the infundibulum (INF), ampulla (A), and isthmus (I). An mRNA of similar size was detected in the oviduct of the sheep, cow, and rabbit, and one of slightly greater size (2.8 kb) in the mouse and hamster oviduct but not in the horse or alligator oviduct. Dot blot analysis indicated that steady-state levels of POSP mRNA were significantly greater (p = 0.0001) in the A than in the INF or I regardless of day of the estrous cycle and were greater on Day 0 (estrus; p = 0.0001) regardless of location. Further, steady-state mRNA levels were significantly increased (p = 0.02) on Days 0 and 1, declining rapidly to Day 2 through Day 15 of the estrous cycle. Steady-state POSP mRNA levels were significantly greater (p < 0.003) in ovariectomized gilts treated with estradiol valerate than those treated with other steroid regimens, vehicle, or no treatment (Control), consistent with estrogen control of mRNA expression. The POSP protein exhibited significant identity to oviductal glycoproteins from the baboon, cow, hamster, human, mouse, and sheep, to several mammalian nonoviductal glycoproteins; and to several chitinases. POSP joins a growing subfamily of the chitinase gene family that lacks chitinase enzymatic activity.
Subject(s)
Cloning, Molecular , Estrogens/pharmacology , Glycoproteins/genetics , Swine , Alligators and Crocodiles , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cricetinae , DNA, Complementary/chemistry , Female , Glycoproteins/chemistry , Humans , Mice , Mice, Inbred ICR , Molecular Sequence Data , Ovariectomy , Protein Biosynthesis , RNA, Messenger/analysis , Rabbits , Sequence Homology , Sheep , Species Specificity , Tissue DistributionABSTRACT
Melatonin is thought to play a role in relaying photic information to the central nervous system as part of the seasonal reproductive cycle of the mare. However, the mechanisms by which melatonin may act are unknown. Therefore, this study was designed to determine whether exposure to constant light would, by reducing circulating melatonin concentrations, have any effect on hypothalamic gonadotrophin-releasing hormone (GnRH) content and circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Blood samples were collected for 12 h at 15-min intervals from 8 ovariectomized (OVX) pony mares under ambient light conditions (12 h light, 12 h dark, October 6). Animals were then placed under representative ambient light (12 h light, 12 h dark; control, n = 4) or constant light (24 h light, 0 h dark; treatment, n = 4) in light-controlled rooms. Blood samples were collected daily and on experimental Days 14, 21 and 28 samples were collected at 15-min intervals for 12 h for analysis of circulating LH and melatonin (Bleed 2, 3 and 4, respectively). All animals were killed on Day 28 (following Bleed 4) and the hypothalami were collected. Mares exposed to constant light had significantly higher (P less than 0.05) LH concentrations in daily blood samples and showed significantly (P less than 0.05) higher LH concentrations during frequent sampling periods on Days 14 and 21 (Bleeds 2 and 3) compared with control mares. FSH did not differ significantly among groups in the daily samples. GnRH content was 1.5994 +/- 0.325 and 0.9457 +/- 0.193 pg/mg protein (treatment and control respectively).(ABSTRACT TRUNCATED AT 250 WORDS)