Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Induction of parturition in cases of pathological gestation in cattle

One or two injections of 20 to 40 mg dexamethasone (i.m.) induced parturition in 17 out of 27 cows (81 %) with dropsy in the 7th to 9th month of gestation. Although the doses employed were high, induction was slow and relaxation of the genital tract was frequently insufficient. Ten cows with mummified fetuses, which were unresponsive to dexamethasone, delivered between Days 3 to 11 after repeated i.m. injection of DES, in doses ranging from 60 to 800 mg. As a rule, the corpus luteum regressed in all cases prior to the onset of parturition. Rapid regression of the corpus luteum also preceded expulsion of mummified fetuses treated with PGF_2α by intrauterine infusion of 10.5 to 45 mg. In cows carrying a macerated fetus, estrogen induced parturition only if regression of the corpus luteum was obtained. However, prostaglandins always caused regression of corpora lutea. The assumption is made that estrogens stimulate prostaglandin production in intact endometrium, causing luteolysis and, subsequently, parturition. Since with mummification the uterine lining remains unimpaired, while it is severely damaged with maceration, estrogens work in the former, but not in the latter situation, in which PGs can be used with success.     

Prostaglandin F-2 alpha is transferred from the uterus to the ovary in the sheep by lymphatic and blood vascular pathways

[3H]Prostaglandin F-2 alpha (PGF-2 alpha) was infused into a uterine lymphatic vessel or a uterine vein for up to 1 h, or injected into the uterine lumen of anaesthetized non-pregnant sheep 7-15 days after oestrus. After an intraluminal injection, labelled PGF-2 alpha was recovered in uterine lymph and peak radioactivity was reached 50 min after injection. [3H]PGF-2 alpha infused at a constant rate into a uterine lymphatic vessel resulted in a maximum concentration of radioactivity in plasma which was 5.6- and 1.7-fold higher in the adjacent utero-ovarian and ovarian vein, respectively, than in carotid arterial plasma. Estimation of the amount of infusate transferred from a lymphatic into ovarian venous blood gave a value (0.4%) similar to that for transfer from a uterine vein (0.3%). Evidence for local transfer was substantiated by the presence of significantly higher concentrations of 3H-labelled compounds in the ovary and corpus luteum adjacent to the site of intra-lymphatic infusion compared with those in the opposite organs. The concentrations in the adjacent ovary and corpus luteum were significantly greater when an intra-lymphatic rather than intra-uterine vein infusion was adopted. The results show that [3H]PGF-2 alpha is transferred locally from uterine lymphatic vessels into the adjacent ovary, corpus luteum and ovarian vein.     

Source and regulation of 17 alpha-hydroxyprogesterone during baboon pregnancy

The present study determined the source and regulation of 17 alpha-hydroxyprogesterone (17-OHP4) during mid-late baboon pregnancy. Serum 17-OHP4 (ng/ml) in 5 untreated baboons increased from low values at mid-late gestation to a mean (+/- SEM) of 0.49 +/- 0.02 during the final 20 days of gestation. Fetectomy of 5 baboons resulted in serum 17-OHP4 concentrations which declined to and remained at baseline. Serum 17-OHP4 concentrations were 5- to 10-fold greater (P less than 0.001) in the uterine, utero-ovarian, and umbilical veins than peripherally. Apparently the fetal adrenal provides precursors for placental 17-OHP4 formation because the fetal adrenal gland develops delta 5-3 beta-hydroxysteroid dehydrogenase only late in gestation, and because the fetal adrenal and not the placenta has the capacity for 17-hydroxylation. Thus, at mid-late gestation the placenta appears to supply a major, and at term the corpus luteum a minor portion of the total 17-OHP4. Administration of the estrogen antagonist ethamoxytriphetol (MER-25, 15 mg/kg BW) to 4 baboons did not affect 17-OHP4 during mid-late gestation, when the placenta was the only source of 17-OHP4. However, MER-25 resulted in serum 17-OHP4 concentrations (ng/ml) at term which were greater (1.08 +/- 0.10, P less than 0.001) than in untreated baboons (0.49 +/- 0.02). Prior removal of the corpus luteum of pregnancy in 4 animals subsequently given MER-25 prevented this rise in 17-OHP4. This suggests that the marked elevation in 17-OHP4 observed near term after MER-25 administration was of luteal origin and that antiestrogen enhanced 17-OHP4 secretion by the corpus luteum.     

In vitro PGF2alpha production by endometrium and corpus luteum explants from pregnant and nonpregnant diestrus bitches and placental explants from pregnant bitches

To better understand the process of slow luteal regression of the nonpregnant cycle in dogs and the acute luteolysis that occurs prepartum, the present study investigated in vitro PGF2alpha production by the endometrium, corpus luteum and placental explants obtained at known times of the cycle from pregnant bitches (days 63, 64 and immediately postpartum; day 0 = estimated day of the ovulatory LH surge) and from nonpregnant diestrus bitches (approximately days 65, 75 and 85). Both basal PGF2alpha production and its production in the presence of the protein kinase C (PKC) stimulator 12,13-phorbol dibutyrate (PDBu) were determined. For PDBu-supplemented incubations, mean PGF2alpha production (pg/mL/mg/6 h) by endometrium explants of the nonpregnant bitches in late diestrus was highest on day 65 (205 +/- 87) and reduced to low levels (38 +/- 17 and 11 +/- 11) on days 75 and 85, respectively. The production by corpus luteum explants from these bitches was significantly less on day 65 (46 +/- 14) than that of the day 65 endometrium explants, and was slightly increased on day 85 (103 +/- 52). The corresponding mean PGF2alpha production by the endometrium explants of pregnant bitches was on average much greater (i.e., two to three-fold) compared to nonpregnant bitches (P < 0.01) and involved high concentrations at day 64 (1523 +/- 467) and postpartum, compared to somewhat lower levels on day 63 (830+/-65); luteal PGF production (165 +/- 4) was also higher than in nonpregnant bitches around day 65. For pregnant bitches, PGF production per gram of tissue in the endometrium explants was greater than for the CL or placenta explants (180 +/- 37). Therefore, the endometrium of the pregnant bitch has an increased capability to produce PGF2alpha immediately prepartum, which on a tissue weight basis, exceeds that of either corpora lutea or the placenta. However, assuming a larger mass of placental tissue in vivo, we inferred that the placenta may contribute substantially to peripheral PGF concentrations.     

Discriminating analysis of "in vitro" prostaglandin release by myometrial and luminal sides of the ewe endometrium

An original perifusion device which allows a discrimination between the 30 mn releases of prostaglandins F2 alpha and E2 by the luminal and the myometrial faces of sheep endometrium is described. Tissue was sampled on day 4, 14, 16 or 17 of the cycle and on day 14 or 17 of pregnancy. Total prostaglandin (PG) release measured with this device was in good agreement with PG's concentrations in media of in vitro endometrium incubations already described. Discrimination analysis of the PGs release by each side of the endometrial tissue during the 30 mn perifusion time revealed that PGF2 alpha concentrations of the perifusion medium issued from the lumen compartment were higher than those of the myometrial compartment in all physiological status where corpus luteum is active (including early pregnancy). Therefore in the ewe, it seems that luteal structure maintenance during early pregnancy is not due, as in the gilt, to a shift in PGF2 alpha secretion towards the uterine lumen.     

The effect of relaxin on the oxytocin receptor in human uterine smooth muscle cells

Experimental objectives: Activation of the oxytocin receptor (OTR) induces phospholipase C induced PIP(2) turnover in the human uterus. Relaxin (RLX), a polypeptide hormone produced in the corpus luteum of pregnancy as well as in the placenta and decidua inhibits PIP(2) turnover and subsequent signaling in human myometrium. The purpose of this study was to evaluate a possible effect of RLX on OTR regulation in human uterine smooth muscle cells. Primary cultures of myometrium from term pregnant women undergoing elective caesarean section were incubated for different time periods (0-96 h) and with different concentrations of RLX [10 pg/ml-20 microg/ml]. The effects on OTR binding, mRNA and protein expression were evaluated by means of (125)I-OVT binding assay, RT-PCR and flow cytometry. RESULTS: Prolonged RLX incubation was able to inhibit 30-40% of OTR binding while binding affinity remained unchanged. Oxytocin receptor mRNA and protein expression were down regulated by RLX about 50% and 35% respectively. CONCLUSION: We report for the first time an effect of RLX on OTR regulation in human uterine myometrial cells. The above results indicate that high local uterine RLX concentrations may be involved in uterine quiescence during human pregnancy by down regulating the OTR.     

Growth and in-vitro metabolism of placental tissues of cows from day 100 to day 250 of gestation.

Weight of placental tissues of cows increased exponentially from Day 100 to Day 250 of gestation, but at much slower relative and absolute rates than fetal weight. In addition, growth rate of fetal placental tissues was less than that of maternal placental tissues. Concentrations of DNA, RNA and protein, however, increased in fetal placental but not in maternal placental tissues. Fetal placental tissues therefore exhibited hyperplasia, which probably contributes to increased functional capacity of the placenta during late gestation. The rate of O2 uptake in vitro was greatest for maternal placental tissues, suggesting that the maternal portion of the placenta accounts for most of the large rate of placental O2 utilization in vivo. Compared with other placental tissues, rate of secretion of macromolecules by intercaruncular endometrium was high, but decreased from Day 100 to 250, suggesting that uterine glandular secretory activity may decrease as gestation advances. Rate of secretion of macromolecules also was high for intercotyledonary tissues and increased with day of gestation, suggesting a role for secretory products of chorioallantois in gravid uterine function.     

Placentation in the opossum, Didelphis virginiana

For the first 9 days of gestation, opossum embryos float in uterine secretions, separated from maternal tissues by a shell membrane. Each embryo is part of the wall of its hollow embryonic sphere. By the 10th day of development, the embryo becomes enveloped by both the amnion and yolk-sac. The yolk-sac consists of vascular and non-vascular portions and, together with the surrounding trophectoderm (trophoblast), forms the yolk-sac placenta of the opossum: the allantois does not contribute to formation of the placenta. The vascular portion of the yolk-sac placenta establishes an intimate relationship with the uterine epithelium soon after loss of the shell membrane. The yolk-sac placenta is non-invasive. Cells of the trophoblast exhibit numerous microvilli, an apical endocytic complex and the lateral and basal cell membrane are elaborately folded. These features suggest a cell that is active in the transport of materials. Junctional complexes between cells of the trophoblast and uterine epithelium were not observed. The uterine epithelium changes from ciliated pseudostratified columnar with few infoldings of lateral and basal cell membranes, to non-ciliated simple columnar in which these membranes show elaborate infoldings. The cells show numerous inclusions and mitochondria are polarized to the basal half of the cell. These features suggest a cell that also is active in the transport of materials.     

Relationship between uterine morphology and peripheral concentrations of sex steroid hormone in wild Japanese black bears (Ursus thibetanus japonicus)

Developing a better understanding of the reproductive physiology and breeding condition peculiar to wild Japanese black bears (Ursus thibetanus japonicus) is crucial for estimation of their habitat distribution. The aim of this study was to clarify the changes in morphology of the genital organs, cellular proliferation in the endometrium and sex steroid hormone concentrations along with the reproductive cycle in Japanese black bears. Samples were collected from a total of 24 female Japanese black bears (1-15 presumptive years old) that were caught in the wild in Iwate prefecture during the period between August 1999 and September 2005. Twenty-two out of the 24 animals were hunted from May to October. The ovaries from the 24 animals and the uteri from 23 animals were observed macroscopically and histologically to examine the relationship between morphology of the genital organs and the month of the year the animal was caught. The staining pattern of proliferating cell nuclear antigen (PCNA) in the endometrium was characterised. Peripheral concentrations of oestradiol-17beta and progesterone were determined by radioimmunoassay. All the animals that had a corpus luteum (n=12) were captured from August to October. The thickness of the endometrium in the animals captured from August to October (n=16) was significantly greater than those in animals captured from May to July (n=5) (P<0.05). From August to October, the thickness of the endometrium and the ratio of the area of the uterine glands to the area of the endometrium in the animals with a corpus luteum (n=12) were significantly greater than those without a corpus luteum (n=4) (P<0.01). Positive PCNA staining was only observed in the uteri of two animals captured in May. There was a significantly positive correlation between plasma progesterone concentrations and the thickness of the endometrium (rho=0.589, P<0.05). There were also significantly positive correlations between the progesterone to oestradiol-17beta ratio (P4/E2 ratio) and the thickness of the endometrium (rho=0.710, P<0.05), and between the P4/E2 ratio and the ratio of the uterine gland area in the endometrium (rho=0.626, P<0.01). These data suggest that the corpus luteum is formed during or just after the breeding season and that the cells in the endometrium and the uterine glands, which proliferate in the early breeding season, grow and develop under the influence of progesterone and oestradiol-17beta during the period of delayed implantation in Japanese black bears.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discriminating analysis of “in vitro” prostaglandin release by myometrial and luminal sides of the ewe endometrium

An original perifusion device which allows a discrimination between the 30 mn releases of prostaglandins F₂α and E₂ by the luminal and the myometrial faces of sheep endometrium is described. Tissue was sampled on day 4, 14, 16 or 17 of the cycle and on day 14 or 17 of pregnancy. Total prostaglandin (PG) release measured with this device was in good agreement with PG's concentrations in media of in vitro endometrium incubations already described.Discrimination analysis of the PGs release by each side of the endometrial tissue during the 30 mn perifusion time releaved that PGF₂α concentrations of the perifusion medium issued from the lumen compartment were higher than those of the myometrial compartment in all physiological status where corpus luteum is active (including early pregnancy). Therefore in the ewe, it seems that luteal structure maintenance during early pregnancy is not due, as in the giltm to a shift in PGF₂α secretion towards the uterine lumen.     

Progesterone values in monkeys near term

The serum progesterone of peripheral, ovarian, uterine and umbilical blood from six Macaca mulatta and two M. fascicularis was determined by radioimmunoassay in late pregnancy. Peripheral progesterone values fell to lower levels the day after delivery, a decrease indicating placental progesterone secretion. The concentration of progesterone in the ovarian vein associated with the presence of a corpus luteum was greater than that in the contralateral vein, a difference denoting active progesterone secretion by the corpus luteum. In most cases the uterine vein on the side associated with the placement of the primary and secondary placentae contained more progesterone than its opposite counterpart, a condition that suggests some synthesis of progesterone by the placenta. The umbilical vein/artery progesterone ratio showed fetal metabolism of this steroid and also greater metabolism of progesterone by the female fetus. The progesterone concentration in the ovarian vein associated with the corpus luteum in mothers who bore female fetuses was greater than that of the same vein in those mothers who bore male fetuses. Peripheral progesterone levels were high the day before cesarean section and fell to lower levels the day after delivery. The decline was more rapid in mothers who bore male fetuses.     

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.     

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.