The recently cloned GnRH receptor, a G-protein coupled receptor that spans the membrane seven times, plays a central role in the maintenance of normal reproductive events. In pituitary gonadotrophs, activation of the GnRH receptor stimulates a concert of intracellular signalling pathways. Phospholipase C stimulation generates inositol 1,4,5 trisphosphate and diacylglycerol, which release calcium and activate protein kinase C, respectively. After these primary signals, prolonged activation of protein kinase C arises from the continued production of diacylglycerol from additional signal transduction pathways. While characteristic calcium responses, involving specific calcium pools, are instrumental in triggering exocytosis, the precise role of protein kinase C activation is unclear. Key players within the exocytotic machinery are also elusive but may include a range of membrane, guanine nucleotide and calcium-binding proteins, inositol 1,4,5 trisphosphate receptors and the cell cytoskeleton. Cellular signalling is also important in determining pituitary responsiveness to GnRH, involving intracellular cross-talk between the GnRH, oestradiol and progesterone receptors.
C. Huang, Y. Li and L. L. Anderson
The role of relaxin and oestrogen in the remodelling of connective tissue was investigated by testing collagenase-dispersed cells (3 × 105 cells per well) from the uterine cervix of gilts for relaxin binding and collagen secretion. Relaxin-binding sites on these cells were quantified by specific binding of a saturating dose of 125I-labelled monotyrosyl relaxin at optimal conditions. Oestrogen at doses from 0.4 to 50 ng ml−1 increased relaxin binding in a time- and dose-dependent manner. Scatchard plot analysis exhibited curvilinearity, which suggested two classes of relaxin-binding sites. The addition of relaxin (0, 100, 500 ng ml−1) alone (P < 0.05) or in combination with oestrogen (oestradiol benzoate: 0, 50, 250 ng ml−1) increased protein secretion into the culture medium. Hydroxyproline concentration (as an index of collagen) in the medium was increased (P < 0.05) only in the presence of both relaxin and oestrogen. Actinomycin D (500 ng ml−1) and cycloheximide (500 ng ml−1) inhibited hydroxyproline secretion induced by combined relaxin and oestrogen treatment. Dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl cAMP: 0, 0.1, 1.0, 5.0 mmol l−1) was a potent stimulator of hydroxyproline secretion. These results indicate that relaxin, probably via a cAMP pathway, stimulates hydroxyproline secretion in the presence of oestrogen through a protein- and RNA-synthesis dependent process. Oestrogen plays a role in augmenting the sensitivity of uterine cervical cells to relaxin in the pig.
K. F. DeBOER and L. L. ANDERSON
An IUD is thought to exert a contraceptive effect either by (1) inducing the premature expulsion of embryos, (2) preventing the uterus from accepting the embryo for implantation, or (3) causing the death of the unimplanted embryo. The intrauterine environment of rats with an IUD was tested for embryotoxic effects on embryos introduced into the uterus, using the technique of double transfer of embryos. Exposure of embryos to an IUD-bearing uterus for 2½ to 4 hr resulted in failure to recover 85% of them. Even the few embryos recovered after 2½ to 4 hr showed greatly increased mortality when transplanted into a normal pseudopregnant recipient. Removing the IUD 6, 24, 48 or 72 hr before embryo transfer increased both the recovery and survival rates over those obtained with the IUD in situ. The recovery rates were not equal to those from the controls, however, until 72 hr elapsed between IUD removal and embryo transfer. Embryos were transferred into the IUD-bearing horn of bilaterally ovariectomized rats treated with progesterone. The recovery rate of these embryos was intermediate between that of intact controls and non-ovariectomized, IUD-bearing animals. The survival rate of these embryos, however, was higher than that of the controls. The results of the embryo transfer experiments indicate that an IUD inhibits pregnancy in the rat by directly causing the death of embryos within 4 hr.
L. L. Anderson and R. O. Parker
The distribution and development of pig embryos were determined in relation to the number of embryos and their positions within the uterine horn between Days 14 and 34 after mating. The observed distribution of 1-11 embryos within a uterine horn was highly correlated (r = 0·96) with the theoretical expected distribution. Embryo spacing was uniform regardless of the number of embryos within the horn. Nitrogen content of the embryo in relation to its position within the uterine horn indicated that development was similar for embryos located at the utero-tubal end or cervical end and comparable to those located in the middle portion of the horn. Placental development, as indicated by nitrogen content, was similar regardless of location within the horn.
B. Bagna, C. Schwabe and L. L. Anderson
Summary. Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2mg, 12 h apart; Group 2R, N = 7). Intervals (mean ± s.e.) between the first injection of relaxin or PBS and calving were 64 ± 17, 80 ± 19 and 125 ± 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P < 0·01) and 1R (P < 0·05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P < 0·01). Body weights and ratios of males to females of the calves were similar (P > 0·05) between groups. Progesterone plasma concentrations decreased (P < 0·01) earlier in Groups IR and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2·7 ± 1·1 ng/ml for Group 2R, 3·5 ± 0·9 ng/ml for Group 1R, and 6·0 ± 0·1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 ± 2·2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0·3 ± 0·01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.
Keywords: relaxin; parturition; progesterone; cervix; pelvic area; dystocia; dairy cattle
L. L. ANDERSON, J. J. FORD and R.M. MELAMPY
After mating, rats were fed one of the following diets: 100% sucrose, 100% vitamin-free casein, purified protein-free or purified with 20% casein. Fetal survival and weight changes in the maternal adrenal glands, uterus, ovaries and gastrocnemius muscle were determined at Day 20. Pregnancy failed in all animals fed sucrose or a protein-free diet and in half of those fed purified casein. Daily injections of progesterone (5 mg) increased both the number of rats remaining pregnant and fetal survival rates in those fed sucrose, a protein-free diet or casein. Pregnancy maintenance in these animals was characterized by larger adrenal glands and smaller gastrocnemius weights. The failure of pregnancy in rats fed only vitamin-free casein or a protein-free diet was associated with a significant increase in loss of total body weight. The minimum period of progesterone treatment for the maintenance of pregnancy was Days 5 to 9 in dams fed only sucrose after mating. In animals fed sucrose, treated with progesterone from Days 5 to 9 and killed on Days 6, 9, 12, 15 and 18, the weights of the uterus and conceptuses began to increase, the weight of the liver declined, followed by a decrease in gastrocnemius weight. Exogenous ACTH or prolactin was inadequate for maintaining viable embryos in ovariectomized rats given a diet of sucrose.
K. F. DeBOER, L. L. ANDERSON and R. M. MELAMPY
The mode of action of intra-uterine devices (IUDs) has been postulated by several investigators to involve a pharmacologically active substance capable of inhibiting the uterine decidual response or of actively cytolysing the pre-implantation blastocysts (Marston & Chang, 1965; Marston & Kelly, 1969; Doyle & Margolis, 1963; Batta & Chaudhury, 1968a, b; Parr, Schaedler & Hirsch, 1967). An investigation of the effect of uterine secretions on ovarian function and embryonic survival in the rat is reported here.
Rats (180 to 250 g; Holtzman Co.) were maintained as previously described (Anderson, Melampy & Chen, 1967). Daily vaginal smears were taken. Day 1 was characterized by the appearance of spermatozoa in mated animals. In the first series of experiments (Table 1), laparotomies were carried out under ether anaesthesia, using clean but not sterile technique,
O. S. Gazal, Y. Li, C. Schwabe and L. L. Anderson
Pregnant ewes were injected with either the antiprogesterone, RU 486 (4 mg kg−1 body weight, i.m.; n = 5), 3000 iu relaxin (i.m.; n = 9), or diluent (n = 8) at 12:00 h on days 144 and 145, to determine its effect on progesterone and relaxin secretion, and on induction of lambing. RU 486 induced earlier lambing (P < 0.01) compared with diluent treatment, but relaxin treatment did not significantly reduce the interval to parturition. Mean injection–lambing intervals were 31 ± 2, 109 ± 23 and 121 ± 27 h for the RU 486, relaxin and diluent groups, respectively. There was no incidence of difficult birth (dystocia); all lambs were vigorous at birth; and placenta delivery was rapid (within 207 min) with RU 486 and relaxin treatments compared with diluent treated controls. Plasma progesterone concentrations averaged 11 ng ml−1 during the pretreatment period for all animals. RU 486 had a biphasic effect on progesterone concentrations, causing an initial increase (P < 0.05) within 2 h, and then an abrupt drop (P < 0.01) to 6 ng ml−1 by 18:00 h on day 145. Progesterone concentrations remained consistently lower (P < 0.05) in relaxin-treated ewes than in diluent-treated controls from days 144 to 147 and then began a steady decrease to 4 ng ml−1 on the day of parturition (days 149 and 150) in both groups. Immunoreactive relaxin concentrations in control ewes increased (P < 0.05) from 0.6 ng ml−1 to a peak of 3.9 ng ml−1 on day 146, but they were low (0.8 ng ml−1) at the time of parturition (day 150). RU 486 treatment abruptly increased (P < 0.05) circulating relaxin concentration, but the amplitude of this antepartum surge was greatly attenuated compared with that of diluent treated controls. Peak RU 486 concentrations in plasma were 7 and 9 ng ml−1 within 2 h after first and second i.m. injection of the hormone, respectively, and stabilized at 4 ng ml−1 at the time of induced lambing (day 145). The results reveal that an antepartum relaxin surge occurs in sheep 4 days before normal parturition (day 150), but that RU 486 greatly attenuates the relaxin surge and abruptly decreases circulating progesterone concentration with an induced parturition (day 145). The results indicate that RU 486 can precisely control the time of parturition in sheep in late pregnancy without detrimental effects of dystocia, retention of placenta or delayed postpartum fertility.
J. R. Molina, A. I. Musah, D. L. Hard and L. L. Anderson
Summary. The middle uterine artery of gilts was occluded unilaterally or bilaterally from Days 25 to 70 after mating. The results showed that vascular occlusion of one (N = 7) or both (N = 6) middle uterine arteries during mid-pregnancy markedly reduced, compared with sham-operated controls (N = 7), development of the conceptuses and decreased peripheral oestrogen (oestrone + oestradiol-17β) concentrations in maternal blood.
L. E. Anderson, M. K. Schultz and M. C. Wiltbank
This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2α, [3H]PGE2, [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2α and [3H]PGD2 was inhibited by non-radioactive PGF2α (IC50 values of 21 and 9 nmol l−1, respectively), PGD2 (35 and 21 nmol l−1), and PGE2 (223 and 81 nmol l−1), but not by PGE1 (> 10 000 and 5616 nmol l−1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l−1) and PGE2 (7 nmol l−1), but minimally by PGD2 (2316 nmol l−1) and PGF2α (595 nmol l−1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2α required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1, PGE2, agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2α, PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.