Pseudopregnant Rabbits Research Articles

Relationship between blood flow and steroidogenesis in the rabbit corpus luteum.

Blood flow in the corpus luteum of the pseudopregnant rabbit was measured with tracer-labelled microspheres before and at 1 and 3 h after saline treatment (N = 8) or after inhibition of progesterone synthesis with aminoglutethimide (N = 10). Before treatment luteal blood flow (29.5 +/- 3.9 ml/min.g-1 (mean +/- s.e.m.] was much higher than blood flow to other tissues (ovarian stroma = 2.9 +/- 0.6; uterus = 0.5 +/- 0.1; adrenal gland = 2.6 +/- 0.2 ml/min.g-1). Aminoglutethimide reduced serum progesterone by 60% within 1 h but luteal blood flow was unchanged (26.2 +/- 3.5 ml/min.g-1). At 3 h after aminoglutethimide, serum progesterone remained low and luteal blood flow was slightly reduced to 22.5 +/- 3.4 ml/min.g-1. This reduction was associated with a significant decline in mean arterial blood pressure which resulted in luteal vascular resistance being unaltered by aminoglutethimide treatment. Further analysis of these data indicated that serum progesterone concentration was not significantly correlated with blood flow to the corpora lutea or with blood flow to other tissues. In contrast, mean arterial blood pressure was highly correlated with blood flow to the corpus luteum (r = 0.80; P less than 0.001) but not to the ovarian stroma (r = 0.04), or adrenal gland (r = 0.06). These results indicate that luteal blood flow is not acutely responsive to changes in luteal progesterone production and suggest that luteal blood flow changes passively with changes in arterial blood pressure.

An immunocytochemical study of the progesterone receptor in rabbit ovary

Distribution of progesterone receptor (PR) was studied immunohistochemically in rabbit ovary and uterus using a monoclonal anti-receptor antibody. At the subcellular level, PR was located in cell nuclei of prepubertal rabbits, which were either non-treated, primed with estrogen or made pseudopregnant. At the tissue level, germinal epithelium, the external theca cell layer and granulosa cell layer were PR-positive. The internal theca cell layer had no PR immunoreactivity. The corpora lutea of pseudopregnant rabbits contained small amounts of PR in some of the animals. During pseudopregnancy, the distribution and staining intensity of PR-positive cells in the ovary was essentially similar to that in non-treated rabbits except for weak PR immunoreactivity in the internal theca cell layer. This differed significantly from the uterus, in which pseudopregnancy caused a marked decrease in PR immunoreactivity. This implies that receptor downregulation by endogenous progesterone is not the same in different organs and cell types. Immunohistochemical techniques give valuable information as to the steroid hormone target cell types and their distribution which are not available by conventional steroid-binding assays.

Platelet-activating factor in the rabbit uterus during early pregnancy.

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.

Pulsatile patterns of gonadotropins and ovarian steroids during estrus and pseudopregnancy in the rabbit.

Consecutive daily plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17 beta (E2), progesterone (P4) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) were monitored in estrous rabbits and in these same doses during pseudopregnancy (PSP); these daily hormone levels, as well as the immediate post-coital changes in gonadotropin secretion, were similar to those in previous reports. To examine the pulsatile patterns of the gonadotropins and ovarian steroids, sequential, 10-min plasma samples were collected for 6 h from estrous does and on Days 3, 10, and 17 of PSP. All five hormones were measured in the serial samples from estrous and PSP Day 10 does; LH and FSH only were assayed in the remaining sequential samples. The amplitude and frequency of FSH pulses did not differ between any of these stages. In marked contrast, LH pulse amplitudes, and even pulse frequencies in Day 17 does, were profoundly increased during PSP above those in estrous does. Progestin secretions, both P4 and 20 alpha-OHP, also were sharply elevated in PSP Day 10 does as compared with those in estrous rabbits; the pulse amplitudes of both progestins were severalfold higher during PSP. P4 pulse frequencies were also increased at this time. Conversely, the parameters of E2 secretion did not differ between estrous and PSP Day 10 animals. In PSP Day 10 does, high amplitude pulses of both P4 and 20 alpha-OHP occurred simultaneously with high amplitude LH pulses. Simultaneous E2 and P4 pulses were evident in these same sequential plasma samples, and this E2-P4 pulse association was greater than that of 20 alpha-OHP pulses with E2 pulses. Our findings failed to identify conclusively the trophic stimulus for the progestin pulse patterns, but the mechanism may involve the coordinated action of LH and E2. The results do demonstrate that each gonadotropin and ovarian steroid is secreted in a pulsatile manner in both estrous and pseudopregnant rabbits. There are altered profiles in LH and progestin pulses, without major changes in FSH and E2 patterns, between the stages of estrus and PSP. The causes and consequences of these divergent endocrine shifts cannot be deduced from these data.

Development of centrifuged cow zygotes cultured in rabbit oviducts.

Zygotes from superovulated cows were centrifuged and pronuclei were detected by differential interference-contrast microscopy in 73% of 106 zygotes. Zygotes were then transferred to ligated oviducts of follicular-phase, 1-day pseudopregnant or 7-day pseudopregnant rabbits and recovered 5 days later. Their development did not differ from that of uncentrifuged zygotes transferred to the opposite oviduct: 41% of the embryos recovered from rabbit oviducts contained 17-32 nuclei and an additional 5% contained greater than 32 nuclei. In another experiment, 399 ova from unmated cows were transferred to rabbit oviducts to determine whether centrifugation induced parthenogenetic development. After 7 days, 257 ova were recovered; 16% of the recovered ova had developed parthenogenetically and contained 2-30 nuclei. Neither centrifugation of the ova nor reproductive status of the rabbits influenced the proportion of parthenogenotes found. Parthenogenetic development was also observed in 14 of 71 ova (20%) recovered on Day 7 from uninseminated superovulated cows. In an attempt to increase the probability of detecting treatment differences, centrifuged and control cow zygotes were incubated for 7 (rather than 5) days in opposite oviducts of fourteen 1-day pseudopregnant rabbits. Development was unaffected by centrifugation: 61% of the zygotes recovered had developed beyond the 16-cell stage, with 23, 24 and 15% containing 17-32, 33-64, and greater than 64 nuclei, respectively. Taking into account the percentage of zygotes in which pronuclei can be seen, the recovery rate from rabbit oviducts, and the proportion of embryos that develop to the morula stage or beyond, 26% of the original group of zygotes would be candidates for transfer into recipient cows.

Gap junction formation in rabbit uterine epithelium in response to embryo recognition

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst.

Isolation and characterization of the specific peptides from rabbit blastocyst at peri-implantation stage with emphasis on their physiological significance relevant to the mechanism of implantation.

The secretory materials from the uterine flushings of the pregnant, non-pregnant and pseudopregnant rabbits were analysed by the method of SDS-polyacrylamide gel electrophoresis. The results demonstrated that there are three specific peptides in the pregnant uterine flushings with molecular weights of 3000, 4500 and 6000. No correspondent peptides were found in non-pregnant and pseudopregnant uterine flushings. The peptides were also isolated and identified from the blastocystic fluid. At least 4 peaks of small peptides were shown by high pressure liquid chromatography. The range of their isoelectrophoretic points is between pH 4.0-5.5 Concavalin A (Con A) affinitive electrophoresis indicated that they are glycopeptides in nature. The LH-RH A is able to exert inhibitory effects on the synthesis of these peptides. Their possible physiological function has been discussed.

Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit.

The intracellular localization of uteroglobin, a progesterone-induced protein, was studied in uterus and oviduct by means of immunoelectron microscopy with the protein A-gold technique. In the uterus, uteroglobin was synthesized in the columnar epithelium of the endometrium where most of the cells were immunoreactive. The protein was localized mainly in small secretory granules which were seen in the process of release into the uterine lumen. The luminal microvilli were also heavily stained. In the oviduct, the secretory cells contained large immunoreactive granules at the apical zone, some of which were observed while discharging into the lumen. Within these secretory granules, uteroglobin accumulated particularly in lens-shaped patches at the periphery of the granules. In vitro kinetic studies on the secretion of newly synthesized uteroglobin indicated that the ability to store uteroglobin is greater in the oviduct than in the uterus; however, the rate of uteroglobin secretion is greater in the uterus than in the oviduct. Thus, there appears to be a good correlation between the microscopic and the functional observations.

Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium.

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.

Evidence for the preferential utilization of esterified cholesterol in progesterone production by rabbit corpora lutea.

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.

Evidence for multiple uterine modulators of rabbit luteal function: the effect of hysterectomy during pseudopregnancy in the estrogen-treated rabbit.

Previous studies show that hysterectomy on Day 1 of pseudopregnancy prolongs serum progesterone secretion in estrogen-treated pseudopregnant rabbits. These studies were undertaken to determine the day of pseudopregnancy when uterine factors are released to alter luteal function. When hysterectomies were performed on either Day 5, 8, 10, or 13 of pseudopregnancy, serum progesterone concentrations were greater than 10 ng/ml between Days 18 and 27 of pseudopregnancy compared to levels of approximately 4 ng/ml in sham-hysterectomized rabbits on these same days. In contrast, serum progesterone levels were not elevated when hysterectomies were performed on Day 11 of pseudopregnancy and were only partially maintained when hysterectomies were performed on Day 12 of pseudopregnancy. Twice daily injections of prolactin (1.5 mg, s.c.) between Days 1 and 33 of pseudopregnancy were unable to mimic the effect of estradiol in the hysterectomized rabbit. Twice daily injections of indomethacin (8 mg/kg, s.c.) between Days 6 and 23 of pseudopregnancy lowered uterine and luteal prostaglandin F2 alpha levels approximately 10-fold on Day 24 of pseudopregnancy but did not maintain progesterone secretion. Serum cholesterol levels were not altered by hysterectomy on any day and were thus not related to the maintenance of progesterone production. These results suggest that the uterus produces both inhibitory and stimulatory factors that effect luteal progesterone secretion. First, an inhibitor is released between Days 10 and 11 of pseudopregnancy in estrogen-treated rabbits that prevents the rabbit corpus luteum from responding to estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation with LH of progesterone production by rabbit corpora lutea in vitro.

Quartered CL from 7-day pseudopregnant rabbits were incubated at 37 degrees C for 0-180 min in the presence of BSA, LH or adrenaline in Krebs-Ringer-bicarbonate buffer. Total progesterone at each time point was quantified in homogenates of tissue plus incubation media and expressed relative to CL protein. Progesterone increased linearly with time during the first 30 min of incubation in the presence of BSA. LH and adrenaline markedly accelerated progesterone accumulations relative to the BSA control. At 10 min, progesterone accumulation in the presence of LH and adrenaline were 2.4 and 5.9 times that in the absence of stimulators, respectively. Both hormones caused concentration-dependent increases in progesterone and the apparent ED50 was 0.75 microgram/ml for LH and adrenaline. The CL obtained from ovaries of 7-day pseudopregnant rabbits are therefore capable of an acute steroidogenic synthetic response to LH as well as adrenaline.

Preparation and characterization of rabbit myometrial cells in primary culture: influence of estradiol and progesterone treatment.

Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits. Isolated cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC) at a plating density of 0.5 X 10(6) cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent cultures. The addition of increasing doses of 17 beta-estradiol (E2) (0.1 nM to 1 microM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E2 (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E2 and P they should provide a useful model to study the regulation of myometrial contractility.

In vitro fertilization with normal development in the sheep.

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two- to six-cell stage within 40 h (75.8% for ovulated and 62.6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20-22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61.9%) and 10 (66.6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained (greater than 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress (greater than 3 months).

Uteroglobin production in the pseudopregnant rabbit uterus. Immunohistochemical studies.

Uteroglobin, a secretory protein of rabbit uterine epithelium, was localized by the direct immunoperoxidase method in control and hCG-induced pseudopregnant rabbits. In control rabbits, uteroglobin was confined to the apical cytoplasm of nearly all cells of the endometrial epithelium. The induction of pseudopregnancy resulted in a pronounced continuing increase, through 4 days post-hCG administration, in the absolute number of epithelial cells engaged in uteroglobin synthesis. Furthermore, the endoplasmic reticulum was more intensely stained for uteroglobin than in the epithelial cells of control rabbit endometrium. Thus, the increased production of uteroglobin, in response to hormonal stimulation, appears to be achieved both through an increase in the amount of uteroglobin synthesized by a given cell as well as by an increase in the number of cells involved in uteroglobin synthesis. Concurrent with the increase in the number of cells synthesizing uteroglobin, an increase in the number of unstained cells first appeared at the second day of pseudopregnancy, during the period of maximal epithelial proliferation. However, within those cells containing uteroglobin on the second day following injection with hCG, most staining was limited to the perinuclear membrane. At various times following hCG administration, a number of scattered cells, intensely stained for uteroglobin, were observed in the uterine epithelium. Based upon ultrastructural studies, failure to exclude trypan blue, and the presence of intra-mitochondrial uteroglobin, they were identified as either dead or dying cells.

Rabbit endocervical epithelium: morphometric analysis of secretory cell populations.

In this report we quantitated ultrastructural changes in two cytologically distinct secretory cell populations from the rabbit endocervix. Type I and type II cells from estrous animals differ only in the presence of one or more empty cytoplasmic vacuoles in type II cells. Comparing type II cells from 5-day pseudopregnant (PSP) rabbits with type II cells from estrous controls, there is no increase (P greater than .05) in the average vacuole volume. When type I and type II cells from PSP animals are compared to cells from estrous controls, there is a decrease (P less than .01) in the average cell volume, a decrease (P less than .01) in the average nuclear volume, and a decrease (P less than .01) in the average granule volume. This reduction in the granule content of secretory endocervical cells was correlated with a dramatic decrease in protein glycosylation into the microsomal fraction. Serum estradiol concentrations for estrous (13.7 +/- 1.0 pg/ml) and PSP (18.1 +/- 1.5 pg/ml) animals were comparable. However, the 36-fold increase in serum progesterone concentrations for PSP (12.04 +/- 1.7 ng/ml) animals compared to estrous (0.33 +/- 0.1 ng/ml) animals may be responsible for the decrease in protein glycosylation.

Cholesterol metabolism in estrogen-sensitive progestin synthesis by rabbit corpus luteum.

To learn whether either reduced de novo cholesterol synthesis and/or altered cholesteryl ester metabolism is responsible for the deficient progestin production induced by estrogen withdrawal from pseudopregnant rabbits, we measured the luteal activity of three enzymes: 1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (the rate-limiting step in de novo cholesterol synthesis), 2) cholesteryl ester hydrolase, and 3) acyl coenzyme A:cholesterol acyltransferase (ACAT) in estrogen-stimulated and estrogen-deprived rabbits. The only change in the activity of these enzymes and of the enzyme NADPH-cytochrome c reductase (a microsomal marker enzyme) after estrogen capsule removal for 12 or 24 h was a 30% decrease in HMG-CoA reductase activity after 24 h. The decrease in HMG-CoA reductase activity was not accompanied by a detectable change in either the content or localization of cellular free cholesterol. Previous data from our laboratory have demonstrated that 24 h of estrogen deprivation has no effect on inner mitochondrial membrane P-450 side-chain cleavage activity (a rate-limiting step in the conversion of cholesterol to steroid hormones). These data, and our earlier finding that estrogen deprivation leads to accumulation of cholesteryl ester in the luteal cells, indicate that estrogen maintains rabbit luteal progestin production by stimulating the transfer of cytoplasmic cholesterol to the active site of P-450 side-chain cleavage on the inner mitochondrial membrane.

Estradiol suppression of luteinizing hormone (LH)/human chorionic gonadotropin receptors and LH-sensitive adenylyl cyclase without decreased adenosine 3',5'-monophosphate content in rabbit corpora lutea.

It has been observed that elevated concentrations of estradiol, the principle luteotropin in the rabbit, reduce LH-stimulated adenylyl cyclase activity in corpora lutea during midpseudopregnancy without suppressing serum and tissue progesterone concentrations. If LH modulates intraluteal cAMP levels in this species, this suggests that in the presence of exogenous estradiol, progesterone synthesis may be independent of cAMP. To test this possibility and to investigate the physiological significance of LH in regulating the rabbit corpus luteum, the concentrations of cAMP and progesterone and the activity of adenylyl cyclase were measured in luteal homogenates, the numbers of LH/hCG receptors were estimated in crude membrane preparations, and the concentrations of progesterone and estradiol were measured in serum on days 2-12 of pseudopregnancy in rabbits treated with or without estradiol. Throughout days 2-12 of pseudopregnancy, estradiol treatment increased LH-stimulated adenylyl cyclase activity on day 2, but reduced its activity after day 4 by up to 50%, reduced the number of LH/hCG receptors after day 2 by up to 50%, and had no effect on the activities of basal, epinephrine-stimulated, or NaF-stimulated adenylyl cyclase. Tissue cAMP levels were not altered by estradiol treatment, nor were serum progesterone concentrations (except for an increase on day 2). Since LH receptors and LH-stimulated adenylyl cyclase were both reduced by day 5 of pseudopregnancy without a concomitant decrease in luteal cAMP or serum progesterone concentrations, our data suggest that LH is not a physiological regulator of luteal cAMP or serum progesterone during days 4-12 of pseudopregnancy. We propose that basal adenylyl cyclase activity, which is not reduced by estradiol treatment, may play a more significant role than LH-stimulated adenylyl cyclase activity in regulating tissue cAMP levels and the progesterone synthetic capacity of the rabbit corpus luteum.

Stage-specific alterations in the apical membrane glycoproteins of endometrial epithelial cells related to implantation in rabbits.

The rabbit endometrial epithelium undergoes differentiation prior to the time of blastocyst implantation, including loss of surface negativity and a change in glycocalyx morphology. Nonpregnant (estrous) and pseudopregnant rabbits were used to study specific alterations in proteins and saccharide composition of the luminal epithelial membrane and its glycocalyx related to the acquisition of receptivity to implantation. Pregnant animals were used to study further modification of the luminal surface by implanting blastocysts. The apical surface of luminal epithelial cells was solubilized by a 15-min intraluminal incubation of 1% Triton X-100 containing protease inhibitors. Proteins in extract solutions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three new polypeptides (24 kDa, 42 kDa and 58 kDa) were identified in uteri from receptive rabbits. Binding of succinyl Wheat Germ Agglutinin (sWGA) and Ricinus communis Agglutinin (RCA-I) lectins to the 24 kDa and 42 kDa components on Western blots of extracts separated by SDS-PAGE identified them as glycoproteins. Additionally, other polypeptides (26 kDa, 80-86 kDa and 145 kDa) showed changes in affinity for WGA, RCA-I or concanavalin A (Con A), depending on the hormonal state. Correlating with these findings was an increased binding of these lectins to intact nonciliated cells in uteri of receptive rabbits compared to estrous animals; ciliated cells bound Dolichos biflorus Agglutinin (DBA) specifically, regardless of the hormonal condition. Treatment of uteri from estrous animals, or Western blots of proteins from these animals, with neuraminidase prior to lectin exposure suggested the presence of glycoproteins having a sialic acid-D-galactose terminus in nonreceptive rabbits. Reduced binding of lectin to intact cells at implantation sites and to blots of proteins isolated from these sites, compared to nonimplantation sites, was noted. These results provide evidence for stage-specific alterations in protein and saccharide composition of the apical surface of endometrial epithelium prior to implantation, and indicate that implanting blastocysts further modify the luminal surface.

Seasonal variations in the responses of luteal and follicular luteinizing hormone-stimulated adenylyl cyclases to estradiol implants and implant removal in pseudopregnant rabbits

1. 1. Summer rabbits appeared to be less sensitive to the presence of exogenous estradiol or to the withdrawal of exogenous estradiol than were Winter rabbits. 2. 2. Suppression of the luteinizing hormone(LH)-stimulated adenylyl cyclase of corpora lutea by estradiol-filled silastic capsules, which resulted in serum estradiol concentrations approximately 3.8 × control (high level), appeared to be greater in Winter rabbits than in Summer rabbits. 3. 3. Both high level and low level (2.1 × control) estradiol implants suppressed follicular LH-stimulated adenylyl cyclase in Winter rabbits but neither size capsule had an effect in Summer rabbits. 4. 4. Serum LH concentrations were equally low in Winter rabbits using either high or low level estradiol implants, while only the high level implants caused a decrease in serum LH in Summer rabbits. 5. 5. Withdrawal of the exogenous estradiol caused a precipitous fall in serum progesterone concentrations in Winter rabbits; estradiol withdrawal in Summer rabbits was without effect.