The in vitro effect of progesterone on the orexin system in porcine uterine tissues during early pregnancy

Background

Orexin A (OXA) and orexin B (OXB) are hypothalamic-derived peptides that participate in the regulation of energy metabolism, food intake and reproductive function by influencing the hypothalamic-pituitary-ovarian axis. Orexins are also produced in the endometrium, myometrium and placenta, which suggests that they could act as a link between energy metabolism and the reproductive system. Changes in the expression of orexin and the orexin receptor genes and proteins during the oestrous cycle and early gestation in pigs imply that orexin activity may be regulated by local factors within the uterus. The aim of this study was to investigate the influence of progesterone (P₄) on the expression of orexin system genes, and proteins in the porcine uterus during early gestation. Gene expression was analyzed by real-time PCR. Adiponectin secretion was determined by ELISA, and the receptors proteins content was defined using western blot analysis.

Results

In the endometrium, P₄ enhanced OXA secretion on days 10 to 11 of gestation and OXB secretion on days 12 to 13. In the myometrium, P₄ inhibited the secretion of both orexins on days 15 to 16 and OXB secretion also on days 12 to 13. In the endometrium, P₄ inhibited the expression of orexin receptor 1 (OX1R) protein at nearly all times analyzed, whereas the expression of orexin receptor 2 (OX2R) protein was inhibited only on days 15 to 16 of gestation. In the myometrium, P₄ stimulated OX1R protein expression on days 12 to 13 and 15 to 16 of gestation and inhibited OX1R protein expression on days 27 to 28. The expression of OX2R protein in the myometrium increased on days 12 to 13 and decreased on days 10 to 11 and 15 to 16.

Conclusions

The results indicate that P₄ could regulate the expression of the orexin system in the porcine uterus during early pregnancy, which suggests the presence of a local feedback loop that could play an important role in the regulation of maternal metabolism during pregnancy. The findings may contribute to the existing knowledge of the mechanisms linking maternal energy metabolism with the regulation of the reproductive system during pregnancy.

     

The effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on endometrial PGF2α synthesis, metabolism and release in early-pregnant pigs

Cytokines produced by the porcine uterus and embryos may be involved in the regulation of endometrial prostaglandin synthesis, metabolism, and release. We studied the effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on: 1) endometrial release of prostaglandin F₂α (PGF₂α), 2) expression of the terminal enzyme of PGF₂α synthesis - PGF synthase mRNA (PGFS mRNA), 3) secretion of PGF₂α metabolite - 13,14-dihydro-15-keto PGF₂α (PGFM) by the endometrium and 4) presence and activity of endometrial NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The effects of cytokines were determined on days 10-11 and days 12-13, e.g., before and during maternal recognition of pregnancy, and on days 15-16, e.g., during the peri-implantation period and compared with its effect in cyclic gilts on corresponding days of the estrous cycle. TNFα did not affect endometrial release of PGF₂α in pregnant and cyclic pigs. IL1β enhanced endometrial PGF₂α release on days 12-13 and 15-16 in pregnant and cyclic pigs, respectively. IL6 increased PGF₂α release mainly on days 15-16 of pregnancy. Expression of PGFS mRNA was decreased by IL1β on days 12-13 of pregnancy (P < 0.05) and increased in response to IL1β, TNFα and IL6 on 12-13 (P < 0.05) and 15-16 (P < 0.01) of the estrous cycle. IL1β increased release of PGFM in gravid pigs on days 12-13, 15-16 and in non-gravid pigs 10-11 and 15-16 of the cycle. On days 15-16 of pregnancy TNFα and IL6 increased endometrial secretion of PGFM. We determined that in porcine endometrium NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is present. In gravid pigs, the highest expression of endometrial 15-PGDH occurred during days 12-13 of pregnancy, while in non-gravid pigs during days 10-11 of the estrous cycle. These data provide new evidence that TNFα, IL1β, IL6 are involved in the regulation of endometrial synthesis, release and metabolism of PGF₂α to protect CL during early pregnancy or to facilitate its regression in cyclic females.     

Changes in plasma orexin A and orexin B concentrations during the estrous cycle of the pig

Orexin A (OXA) and orexin B (OXB) are neuropeptides synthesized mainly in the lateral hypothalamus, which are involved in the control of various physiological functions such as energy homeostasis, sleep, wakefulness and feeding behavior. The present study analyzes orexins A and B levels in the porcine plasma during the estrous cycle. The highest plasma concentrations of orexin A were observed on days 2–3 of the estrous cycle (p < 0.05 relative to days 10–12 and 14–16) and the lowest (p < 0.05) on days 14–16. The highest orexin B levels in the blood plasma were noted on days 17–19 (p < 0.05 vs. days 14–16). We demonstrated the presence of OXA and OXB in porcine blood plasma and the impact of the phase of the estrous cycle on the observed changes in plasma orexin levels.     

A study of prostaglandin F2α as the luteolysin in swine: V comparison of prostaglandin F, progestins, estrone and estradiol in uterine flushings from pregnant and nonpregnant gilts

Uterine flushings were collected from 38 gilts representing Days 6,8,10,12,14,15,16 and 18 of the estrous cycle and pregnancy. The same group of gilts were represented within each of the respective days of the estrous cycle and pregnancy, i.e., three to six gilts per day per status. Uterine flushings (about 40ml) were assayed for prostaglandin F (PGF), estrone (E₁), estradiol (E₂), progestins (P) and protein. Nonpregnant gilts had higher (P<.01) concentrations of P in uterine flushings than pregnant gilts, but pregnant gilts had higher (P<.01) E₁ and E₂ concentrations. Significant day by status interactions were detected for E₁ (P<.05), but not for E₂ concentrations in uterine flushings. Total recoverable PGF and PGF concentrations in uterine flushings were greater (P<.01) in pregnant than nonpregnant gilts and significant (P<.01) day by status interactions were detected. In nonpregnant gilts, PGF increased between Days 12 and 16, i.e., during the period of corpora lutea (CL) regression. In pregnant gilts, PGF in uterine flushings increased markedly between Days 10 and 18. Total recoverable PGF on Day 18 of the estrous cycle was only 464.5 ± 37.6 ng as compared to 22,688.1 ± 1772.4 ng on Day 18 of pregnancy. Total recoverable protein was also higher (P<.01) in pregnant gilts. These data indicate that PGF synthesis and secretion by the uterine endometrium and/or conceptuses is not inhibited during pregnancy and suggest that PGF is sequestered within the uterine lumen of pregnant gilts, as is the total protein component of endometrial secretions referred to as histotroph.     

The effect of interleukin-1β, interleukin-6, and tumor necrosis factor-α on estradiol-17β release in the myometrium: The in vitro study on the pig model

Estradiol-17β (E₂) is a potent regulator of early pregnancy and the estrous cycle in pigs. Production of E₂ occurs in the porcine myometrium, but the factors involved in its regulation are unknown. In this in vitro study, it was investigated whether interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α affect the release of E₂ from the porcine myometrium on Days 10 to 11, 12 to 13, and 15 to 16 of pregnancy and the estrous cycle. The expression of the cytochrome P450 family 19 (CYP19) gene and the presence of the aromatase cytochrome P450 protein in the myometrium confirmed the ability of the tissue to produce E₂. In gravid pigs, the expression of IL1RI mRNA and IL6R mRNA was markedly increased on Days 15 to 16 of gestation, whereas TNFRI mRNA was increased on Days 10 to 11 of gestation. In cyclic pigs, the expression of myometrial IL1RI mRNA did not differ among the studied days, although the expression of IL6R and TNFRI mRNAs was increased on Days 15 to 16. In gravid pigs, IL-1β, IL-6, and TNF-α increased myometrial E₂ secretion on Days 15 to 16 but did not affect E₂ release on Days 10 to 11 and 12 to 13 of pregnancy. In cyclic pigs, IL-1β, IL-6, and TNF-α did not increase myometrial E₂ release. In conclusion, IL-1β, IL-6, and TNF-α affected myometrial E₂ release in a manner that is dependent on the physiologic status of the female. The porcine myometrium expresses IL1RI, IL6R, and TNFRI genes and is the target tissue for IL-1β, IL-6, and TNF-α. In gravid pigs, IL-1β, IL-6, and TNF-α may increase myometrial release of E₂in vitro specifically on Days 15 to 16 of pregnancy. These findings may be of interest to researchers using pigs as an animal model for fetal programming.     

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP – on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) – on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.     

Comparative studies of prostaglandins F2α and E2 in late cyclic and early pregnant sheep: synthesis by endometrium and conceptus effects of indomethacin treatment on establishment of pregnancy

Endometrial concentrations of prostaglandins F₂α (PGF₂α) and E₂ (PGE₂) were measured by specific radioimmunoassay in sheep, on day 14 of estrous cycle or pregnancy, during luteolysis (Day 16 of the cycle), and after implantation (Day 23 of pregnancy) : concentrations observed on day 14 of cycle and pregnancy were similar. During luteolysis, on day 16 of cycle, a consistent drop was noticed. If luteal regression did not occur, as a consequence of the presence of an embryo, endometrial concentrations of PGF₂α on day 23, were twice those of day 14, and PGE₂ remained unchanged. 2 hour incubations of endometrial caruncular tissue from 14 days cyclic or pregnant ewes resulted in de novo synthesis of PG which could be increased by Arachidonic Acid and inhibited by Indomethacin; during the first 30 min of incubation, the PGF₂α synthesis was comparable for both endometrial tissues, whereas PGE₂ synthesis was twice as great in pregnant endometrium. Fourteen and 23 day conceptuses had high PGF₂α and PGE₂ concentrations which were not due to maternal PG sequestration : PG synthesis which could be inhibited by Indomethacin was observed in incubated 14 day old embryos. Treatment of pregnant ewes from day 7 to day 22 after mating, either with Indomethacin (300 mg s.c. daily) or with Acetylsalicylic Acid (1 g I.V. daily) resulted in a sharp diminution of endometrial PG concentration and release, with no apparent effect on the establishment of pregnancy. These results tend to ascribe a less important role to PG during early pregnancy in sheep as compared with rodents, in terms of embryonic growth and implantation.     

Effect of conceptus secretions on HOXA10 and PTGS2 gene expression, and PGE2 release in co-cultured luminal epithelial and stromal cells of the porcine endometrium at the time of early implantation

Homeobox A10 (HOXA10) gene expression was demonstrated in the endometrium of adult porcine uteri, however there is little information concerning the role of this gene in the pig. Objectives of the present study were to examine: 1) the expression of HOXA10 in the endometrium of cyclic and early pregnant gilts; 2) the effect of estradiol (E₂) and progesterone (P₄) on HOXA10 expression in porcine luminal epithelial (LE) and stromal (ST) cells in vitro; 3) the effect of E₂ and conceptus-exposed medium (CEM) on HOXA10 and prostaglandin endoperoxide synthase (PTGS2) gene expression and prostaglandin (PG) E₂ secretion from LE and ST cells in a co-culture model. The abundance of HOXA10 mRNA was increased on day 15 of pregnancy in comparison to day 15 of the estrous cycle. Moreover, increased HOXA10 mRNA level was detected in ST cells after E₂ and P₄ treatment. E₂ stimulated the expression of HOXA10 in LE cells cultured on collagen and pre-treated with steroids, but not in LE on plastic surfaces. Addition of CEM to LE cells cultured in collagen-coated inserts of the co-culture system resulted in elevated HOXA10 and PTGS2 gene expression and PGE₂ secretion in these cells, but not in ST cells cultured in basal compartments. ST cells directly treated with E₂ or CEM showed higher levels of HOXA10 and PTGS2 expression. Blocking of estrogen receptors with ICI-182,780 did not influence the stimulatory effect of CEM. We conclude that HOXA10 expression in the porcine endometrium is closely related to the implantation process and stimulated by conceptus products. Moreover, the co-culture system of LE and ST cells is a promising model for the study of endometrial response to conceptus-derived factors.     

Expression of the orexin system in the porcine uterus,conceptus and trophoblast during early pregnancy

Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.     

Comparative studies of prostaglandins F2α and E2 in late cyclic and early pregnant sheep: In vitro synthesis by endometrium and conceptus effects of in vivo indomethacin treatment on establishment of pregnancy

Endometrial concentrations of prostaglandins F₂α (PGF₂α) and E₂ (PGE₂) were measured by specific radioimmunoassay in sheep, on day 14 of estrous cycle or pregnancy, during luteolysis (Day 16 of the cycle), and after implantation (Day 23 of pregnancy) : concentrations observed on day 14 of cycle and pregnancy were similar. During luteolysis, on day 16 of cycle, a consistent drop was noticed. If luteal regression did not occur, as a consequence of the presence of an embryo, endometrial concentrations of PGF₂α on day 23, were twice those of day 14, and PGE₂ remained unchanged. $\text{In vitro}$ 2 hour incubations of endometrial caruncular tissue from 14 days cyclic or pregnant ewes resulted in de novo synthesis of PG which could be increased by Arachidonic Acid and inhibited by Indomethacin; during the first 30 min of incubation, the PGF₂α synthesis was comparable for both endometrial tissues, whereas PGE₂ synthesis was twice as great in pregnant endometrium. Fourteen and 23 day conceptuses had high PGF₂α and PGE₂ concentrations which were not due to maternal PG sequestration : $\text{de novo}$ PG synthesis which could be inhibited by Indomethacin was observed in incubated 14 day old embryos. Treatment of pregnant ewes from day 7 to day 22 after mating, either with Indomethacin (300 mg s.c. daily) or with Acetylsalicylic Acid (1 g I.V. daily) resulted in a sharp diminution of endometrial PG concentration and release, with no apparent effect on the establishment of pregnancy. These results tend to ascribe a less important role to PG during early pregnancy in sheep as compared with rodents, in terms of embryonic growth and implantation.     

The involvement of peroxisome proliferator activated receptors (PPARs) in prostaglandin F2α production by porcine endometrium

In the present study, we investigated the in vitro effects of peroxisome proliferator activated receptor (PPAR) ligands on PGF_2α secretion and mRNA expression of prostaglandin F synthase (PGFS) in porcine endometrial explants collected on days 10–12 and 14–16 of the estrous cycle or pregnancy. The explants were incubated for 6 h with: PPARα ligands – WY-14643 (agonist) and MK 886 (antagonist); PPARβ ligands – l-165,041 (agonist) and GW 9662 (antagonist); PPARγ ligands – 15d-prostaglandin J₂ (PGJ₂, agonist), rosiglitazone (agonist) and T0070907 (antagonist). The expression of PGFS mRNA in the endometrium and the concentration of PGF_2α in culture media were determined by real time RT-PCR and radioimmunoassay, respectively. During the estrous cycle (days 10–12 and 14–16), the agonists – WY-14643 (PPARα), l-165,041 (PPARβ), PGJ₂ and rosiglitazone (PPARγ) – increased PGF_2α secretion but did not affect PGFS mRNA abundance. During pregnancy (days 10–12 and 14–16), PPARα and PPARγ ligands did not change PGF_2α release, whereas PPARβ agonist augmented PGF_2α release on days 14–16 of pregnancy. In addition, WY-14643 and l-165,041 increased PGFS mRNA level in both examined periods of pregnancy. PPARγ agonist (PGJ₂) and antagonist (T0070907) enhanced PGFS mRNA abundance in the endometrium on days 10–12 and 14–16 of pregnancy, respectively. The results indicate that PPARs are involved in the production of PGF_2α by porcine endometrium, and that the sensitivity of the endometrium to PPAR ligands depends on reproductive status of animals.     

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.     

11-Ketotetranor PGF metabolites,a suitable indicator for measuring prostaglandin release during the normal oestrous cycle and early pregnancy in the goat

Peripheral plasma levels of 15-ketodihydro-PGF_2α, 11-ketotetranor PGF metabolites and progesterone were measured during normal oestrous cycle and early pregnancy in six goats. The does were synchronized before the start of the study by means of 10 mg of PGF_2α. Blood samples were collected twice daily until day 12 of the oestrous cycle and subsequently every 3 h until the onset of oestrus, at which time the does were mated. The blood sampling protocol was repeated until day 28 of pregnancy. High pulsatile peaks of 15-ketodihydro-PGF_2α and 11-ketotetranor PGF metabolites were observed during the last days of the oestrous cycle, indicating PGF_2α releases. This coincided with a fall in progesterone levels. During early pregnancy no such peaks of prostaglandin metabolites were recorded and high levels of progesterone were maintained. In the goat, analysis of the 11-ketotetranor PGF metabolites seems to be a better indicator of PGF_2α release than the analysis of 15-ketodihydro-PGF_2α. The former metabolites are more long-lived in the circulation and are thus easier to detect.     

Absence of direct effects of prostaglandins on rabbit blastocysts in vitro

A total of 27 monkeys (M. Fascicularis) whose control cycle lengths ranged from 28 to 32 days were used in this study. All the treatments described below started either on day 17 or 18 of the cycle. Six monkeys received daily injections of 20 μg estradiol-17β (E₂) for 5 consecutive days. Although a drop in blood progesterone (P) did occur due to this treatment, no shortening of the luteal phase of the cycle was recorded. Seven monkeys received daily injections of 15 mg PGF_2α (prostaglandin-F_2α) for 4 or 5 days. These monkeys also showed a drop in blood P levels; moreover 5 of these monkeys had vaginal bleeding for 2–3 days starting either on day 19 or 20 of the cycle. This bleeding did not appear to be a normal physiological menstrual flow, since all of the monkeys commenced menstrual flow at the expected time. Four monkeys received daily injections of 10 mg P for 3 days. These monkeys also had normal cycle lengths in spite of the treatment. Finally 9 monkeys received daily injections of 20 μg E₂ for 3 days, and starting on the third day of E₂ treatment these monkeys also received injections of 15 mg PGF_2α for 4 or 5 days. Shortened cycle lengths were recorded in 8/9 monkeys in this group. Six monkeys had 22-day cycles, 2 monkeys had 24-day cycles and the remaining monkey had a cycle length of 26 days. Thus 8/9 monkeys had shortened luteal phases due to sequential treatment of E₂ and PGF_2α. The cycle lengths in all the treatment groups were normal subsequent to treatments. These results provide potentially useful information for further studies in the human as a method of contraception.     

Caprine endometrial stromal cells modulate the effects of steroid hormones on cytokine secretion by endometrial epithelial cells in vitro

The main purpose of this study was to examine the effects of 17β-estradiol (E₂) and progesterone (P₄) on cytokine secretion by caprine endometrial epithelial cells (EEC) in vitro. Epithelial cells grown alone or in co-culture with stromal cells (ESC) were treated with E₂ or P₄, or both. Homogeneity of the endometrial cell populations was ascertained immunocytochemically. The quantities of cytokines secreted in this system were assessed by ELISA and their protein expression by Western blot. The exposure of EEC to P₄ alone or in combination with E₂ significantly increased the amount of TGF-β1, TNF-α and IL-18 secretion, whereas E₂ had no effect on the synthesis of these cytokines. When epithelial cells were co-cultured with ESC, the secretion of TGF-β1, TNF-α and IL-18 by EEC significantly increased compared to that by EEC alone. However, the treatment with both steroids decreased the secretion of TNF-α, IL-18 and TGF-β1 by EEC in the presence of ESC. In contrast to TGF-β1, TNF-α and IL-18, the secretion of leukemia inhibitory factor (LIF) by EEC was not affected by E₂ and/or P₄ either directly or indirectly. The present results indicate that the interactions between caprine endometrial stromal and epithelial cells can modulate the secretion of TGF-β1, TNF-α and IL-18 by EEC exposed to E₂ and/or P₄ in vitro.     

Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE(1) and PGE(2)) and F(2alpha) (PGF(2alpha)) by ovine corpora lutea of the estrous cycle or pregnancy in vitro

LH regulates luteal progesterone secretion during the estrous cycle in ewes and cows. However, PGE, not LH, stimulated ovine luteal progesterone secretion in vitro at day 90 of pregnancy and at day 200 in cows. The hypophysis is not obligatory after day 50 nor the ovaries after day 55 to maintain pregnancy in ewes. LH has been reported to regulate ovine placental PGE secretion up to day 50 of pregnancy and by pregnancy-specific protein B (PSPB) after day 50 of pregnancy. The objective of this experiment was to determine if and when a switch from LH to PGE occurred as the luteotropin regulating luteal progesterone secretion during pregnancy in ewes. Ovine luteal tissue slices of the estrous cycle (days 8, 11, 13, and 15) or pregnancy (days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90) were incubated in vitro with vehicle, LH, AA (precursor to PGE(2) and PGF(2alpha) synthesis), or PSPB in M199 for 4 h and 8 h. Concentrations of progesterone in jugular venous plasma of bred ewes increased (P< or =0.05) after day 50 and continued to increase through day 90. Secretion of progesterone by luteal tissue of non-bred ewes on days 8, 11, 13 and 15 and by bred ewes on days 8, 11, 13, 15, 20, 30, 40, and 50 was increased (P< or =0.05) by LH, but not by luteal tissue from pregnant ewes after day 50 (P> or =0.05). LH-stimulated progesterone secretion by luteal tissue from day 15 bred ewes was greater (P< or =0.05) than day 15 luteal tissue from non-bred ewes. Concentrations of progesterone in media were increased (P< or =0.05) when luteal tissue from pregnant ewes on day 50, 60, or 90 were incubated with AA or PSPB. Concentrations of PGE in media of non-bred ewes on days 8, 11, 13, or 15 and bred ewes on days 8 and 11 did not differ (P> or =0.05). Concentrations of PGE were increased (P< or =0.05) in media by luteal slices from bred ewes on days 13, 15, 20, 30, 40, 50, 60, and 90 of vehicle, LH, AA or PSPB-treated ewes. In addition, PSPB increased (P< or =0.05) PGE in media by luteal slices from pregnant ewes only on days 40, 50, 60, and 90. Concentrations of PGF(2alpha) were increased in media (P<0.05) of vehicle, AA, LH, or PSPB-treated luteal tissue from non-bred ewes and bred ewes on day 15 and by luteal tissue from bred ewes on days 20 and 30 after which concentrations of PGF(2alpha) in media declined (P< or =0.05) and did not differ (P> or =0.05) from non-bred or bred ewes on days 8, 11, or 13. It is concluded that LH regulates luteal progesterone secretion during the estrous cycle of non-bred ewes and up to day 50 of pregnancy, while only PGE regulates luteal progresterone secretion by ovine corpora lutea from days 50 to 90 of pregnancy. In addition, PSPB appears to regulate luteal secretion of progesterone from days 50 to 90 of pregnancy through stimulation of PGE secretion by ovine luteal tissue.     

Hormones and sexual behavior associated with postconception perineal swelling in the sooty mangabey (Cercocebus torquatus atys)

Three studies were conducted with female sooty mangabeys to describe the occurrence of perineal swelling during pregnancy and to document the associated patterns of ovarian hormone secretion and sexual behavior. Menstrual cycles and pregnancies were charted in 29 female subjects by recording daily vaginal bleeding and perineal swelling. As in other swelling species, maximum perineal tumescence corresponded with the periovulatory phase of the cycle; however, a second tumescence was recorded during pregnancy, with a peak at 49 days postconception. The swelling patterns were similar with the exception that deturgescence was slower during pregnancy. In a second study, blood samples were obtained weekly from nine females to document the estradiol (E₂) and progesterone (P₄) profiles during pregnancy. Analyses revealed increased E₂ concentrations and low P₄ levels in association with the postconception swelling. A third study compared sexual behavior during ovulatory and post-conception tumescent peaks. Behavioral data revealed that, although there was no overall difference in male mount rate to females during the ovulatory and postconception maximum swelling period, when only the alpha male was considered, significantly fewer mounts were directed to females exhibiting a post-conception versus an ovulatory maximum tumescence.     

Excessive ovarian follicular development in pregnant pigtailed macaques (Macaca nemestrina)

To evaluate the status and possible control of ovarian follicular development during pregnancy, circulating levels of estrone (E₁), estradiol-17β (E₂), and follicle-stimulating hormone (FSH) were measured throughout gestation in both intact and ovariectomized pregnant pigtailed monkeys (Macaca nemestrina). From an additional group of pregnant monkeys, ovaries were obtained at late gestation (on day 150 or 159 of pregnancy) for histological studies. Circulating concentrations of E₁ and E₂ increased on day 13 and remained elevated for about 10 days; they then declined and reached low levels on day 32 of gestation. After day 60, there were gradual but smaller increases in estrogen levels to day 140, after which both E₁ and E₂ levels increased significantly, reaching maximum levels (E₁ = 832.2 ± 210.8 pg/ ml; E₂ = 1.66 ± 0.32 ng/ml) at the end of pregnancy. Removal of ovaries on day 35 of gestation did not affect pregnancy or the pattern of estrogen secretion. Serum concentrations of FSH demonstrated only minor fluctuations during pregnancy but were similar to those found during the early follicular phase of cycling pigtailed monkeys investigated in this study. Ovarian histology revealed extensive follicular growth; in addition to the corpus luteum of pregnancy, ovaries were packed with pre-antral, small antral, and medium-sized Graafian follicles. Some of these follicles appeared to be cystic and showed various degree of atresia; their general appearance was similar to the follicles of human females with polycystic ovary syndrome. Our data suggest that FSH may initiate ovarian follicular growth during gestation. High levels of estrogens were incapable of suppressing FSH secretion but may be responsible for the induction of atresia in a large number of follicles in pregnant pigtailed monkeys.     

Sialic acid binding protein of human endometrium: Its regulation by steroids

In the present study, we have observed that sialic acid binding protein (SABP – a 54 kDa glycoprotein which was isolated from human endometrial scrapings taken at various stages of the menstrual cycle from normal cycling females and purified to apparent homogeneity and was earlier reported from this laboratory) was found in sufficiently detectable amount in the endometrium of normal cycling women whereas it was found in lesser amount in tissue from women who have recently entered the post-menopause stage. SABP was observed in both follicular and luteal phase of menstrual cycle which was found by western blot analysis. In the de-novo synthesis experiment, synthesis and secretion of SABP was found to be stimulated by estradiol (E₂) whereas progesterone (P₄) was found to have no significant stimulatory effect on it which was also confirmed by HEC cell culture. In the HEC cell culture, priming of cells with E₂ was found to influence the effect of P₄ on SABP when it was added 2 h after E₂ administration. This was observed by doing immunoprecipitation followed by SDS-PAGE and autoradiography. Hence this report clearly indicates that E₂ regulates the synthesis and secretion of 54 kDa SABP from human endometrium. How E₂ priming of endometrium influences the effect of P₄ on SABP has been discussed.