Neural regulation of the bovine corpus luteum

The ovarian noradrenergic stimulation or noradrenaline (NA) administration directly to the ovary in cow increases ovarian oxytocin (OT) release and post-translational processing of OT synthesis within a few minutes has been established in both in vivo and in vitro studies. Furthermore, NA affects progesterone secretion and its synthesis by an increase of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase activity. This effect is mediated via luteal cell beta(1)- and beta(2)-receptors. Their total amount correlates with peripheral progesterone concentrations during the luteal phase and this reflects the ability of the ovary to react to beta-stimulation. On the other hand, ovarian denervation causes a decrease of steroidogenic activity in the CL, an increase of beta-receptors on luteal cells, a delay in follicular development and the disruption of cyclicity. Moreover, decrease of progesterone secretion by 20-30% was seen after brief pharmacological blockade of ovarian beta-receptors in the mid-cycle of cattle. We assume that tonic beta-stimulation of the CL ensures the basal secretion of progesterone, whereas acute noradrenergic activation supports the CL during stressful situations which could impair its function. Conversely, long-lasting increase in blood catecholamine concentrations markedly decreases the number of beta-receptors in CL, presumably due to their down-regulation. Concentrations of dopamine (DA) within the CL are highly correlated with those of NA during the estrous cycle, and are higher in the newly-formed than in the developed corpus luteum, the regressed corpus luteum or the corpus luteum of pregnant females. Bovine CL can synthesise de novo NA from DA as a precursor. Concluding, presented data indicate that noradrenergic stimulation can be an important part of mechanism supporting secretory function of CL.     

Characteristics of developing prolonged dominant follicles in cattle

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.     

Immunohistochemical localization of steroidogenic enzymes in the corpus luteum and the placenta of the ribbon seal (Phoca fasciata) and steller sea lion (Eumetopias jubatus)

To study the luteal and placental function of pinnipeds, we analyzed the localization of steroidogenic enzymes (P450scc, 3 beta HSD and P450arom) in the corpus luteum and the placenta of ribbon seals (Phoca fasciata) and Steller sea lions (Eumetopias jubatus) immunohistochemically. P450scc and 3 beta HSD were present in all luteal cells of both species. Almost all of the luteal cells were immunostained for P450arom, while P450scc and 3 beta HSD were negatively immunostained in placentae and P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) corpora lutea of both species synthesize pregnenolone, progesterone and estrogen during the entire pregnancy period, and 2) like other terrestrial carnivores in the suborder Caniformia, placentae of both species do not have the capability for synthesizing progesterone in the latter half of active pregnancy period.     

Oxytocin modulates the pulsatile secretion of prostaglandin F2alpha in initiated luteolysis in cattle

Subluteolytic doses of prostaglandin F2alpha analogue (oestrophan) given i.m. and oxytocin (OT) antagonist (CAP) and noradrenaline (NA) infused into the abdominal aorta were used to test the importance of luteal OT in pulsatile secretion of prostaglandin F2alpha (PGF) during luteolysis in heifers (n = 17). In experiment 1, heifers were pre-infused for 30 minutes with saline on either day 17 of the oestrous cycle (group 1; n = 4) or on day 18 of the oestrous cycle (group 2; n = 3), and with CAP (8 mg per animal) on day 17 of the oestrous cycle (group 3; n = 4). Next, heifers were injected with oestrophan (30 microg per animal). Injection of oestrophan in Group 3 increased OT concentrations (P < 0.001) to values similar to those observed during spontaneous luteolysis (50 to 70 pg ml(-1)). PGFM concentrations in this group also increased (P < 0.001), but were lower (P < 0.05) than the values in groups 1 and 2, CAP given prior to oestrophan decreased both PGFM elevation (P < 0.06) and its area under the curve (P < 0.01), compared to the saline pretreated heifers. In experiment 2 NA (4 mg) was infused twice for 30 minutes at five hour intervals to release OT on day 17 of the oestrous cycle (n = 6). However, during hormone analysis it appeared that three of six heifers had elevated PGFM concentrations (group 1) and three others did not (group 2). NA caused the correlated increase of progesterone and OT secretion (r = 0.68; P < 0.05) in both groups but it only influenced PGF secretion in group 1 only (P < 0.05). We postulate that OT can amplify and modulate the course of induced luteolysis as a regulator of the amplitude of pulsatile PGF secretion. PGF analogue stimulates secretion of endogenous PGF from the uterus in cattle and this may be an important component of the luteolytic response to exogenous PGF.     

Immunohistochemical localization of steroidogenic enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca largha) during late pregnancy

To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3betaHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3betaHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3betaHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.     

Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese Shiba goat

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.     

Neurosteroidogenesis in oligodendrocytes and Purkinje neurones of cerebellar cortex of dogs

The cerebellum is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Purkinje neurones appear to be the most active steroidogenic cells in the cerebellar cortex. These neurones express 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), P450 side-chain cleavage (P450scc), 17 alpha-hydroxylase/c17, 20lyase (P450c17), P450 aromatase (P450arom) and produce pregnenolone, progesterone, dehydroepiandrosterone, androstenedion, oestradion and oestrone. Oligodendrocytes are predominantly the producer of myeline protein. The oligodendrocytes were identified by immunohistochemistry using a monoclonal antibody against myeline 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a myeline specific enzyme. In this study we have examined the distribution of 3 beta-HSD and CNPase by immunohistochemistry using monoclonal antibody in canine cerebellar cortex. The localization of oligodendrocytes within the cerebellar cortex was determined to be close to Purkinje neurones. This result suggests that endogenous progesterone synthesized de novo in the Purkinje neurone can promote myeline protein synthesis in oligodentrocytes.     

Oxytocin stimulates secretion of prostaglandin F(2alpha) from endometrial cells of swine in the presence of progesterone

Oxytocin (OT) stimulates endometrial secretion of prostaglandin (PG) F(2 alpha) during corpus luteum regression in swine but there is differential responsiveness to OT among endometrial cell types. To determine if progesterone influenced responsiveness of luminal epithelial, glandular epithelial, and stromal cells to 100 nM OT during luteolysis in swine, cells were isolated from endometrium of 15 gilts by differential enzymatic digestion and sieve filtration on day 16 postestrus and cultured continuously in the presence of 0, 10 or 100 nM progesterone. For phospholipase C (PLC) activity and PGF(2 alpha) secretion, stromal cells were most responsive to OT (P<0.01) in the absence of progesterone, whereas luminal epithelial cells were unresponsive and glandular epithelial cells displayed an intermediate response to OT (P<0.09). Progesterone enhanced PLC activity linearly in glandular epithelial cells (P<0.05) and influenced it quadratically in stromal cells (P=0.05). The effect of OT and progesterone on PLC activity in luminal epithelial cells was not significant, and progesterone did not increase PLC activity in response to OT in any cell type. Culture in the presence of progesterone, enhanced PGF(2 alpha) secretion in response to OT in luminal epithelial cells (P<0.05) but not in glandular epithelial or stromal cells. Progesterone also increased overall PGF(2 alpha) release from glandular epithelial (P<0.05) and stromal cells (P<0.06) across both levels of OT treatment. These results indicate that progesterone enhanced PGF(2 alpha) secretion from luminal epithelial cells in response to OT and increased basal PGF(2 alpha) release from glandular epithelial and stromal cells.     

Involvement of high-density lipoprotein in stimulatory effect of hormones supporting function of the bovine corpus luteum

The hypothesis that epinephrine (noradrenaline, NA) enhances utilisation of high-density lipoproteins (HDL) by bovine luteal cells and that this process involves phospholipase (PL) C and protein kinase (PK) C intracellular pathway was tested. Luteal cells from days 2-4, 5-10 or 11-17 of the oestrous cycle were preincubated for 20 h. Subsequently DMEM/Ham's F-12 medium was replaced by fresh medium and the cells were treated for 6 h as follows: In Experiment I with HDL (5-75 micrograms cholesterol per ml), NA, isoprenaline (ISO) or luteinising hormone (LH). In Experiment II cells were incubated for further 24 h in deficient medium (without FCS) and next treated as in Experiment I. In Experiment III cells were stimulated with NA, ISO or LH alone and together with HDL. In Experiment IV cells were treated with PLC inhibitor (U-73122) or with PKC inhibitor (staurosporine) or stimulator (phorbol 12-myristrate 13-acetate) and with either NA, insulin or LH. Only luteal cells from days 5-10 of the cycle responded on HDL and beta-mimetics (P < 0.05). LH stimulated progesterone secretion from the luteal cells during all stages of the cycle (P < 0.001). Cells incubated in deficient medium and supplemented with HDL secreted as much progesterone as those stimulated by LH in all stages of the cycle. Beta-mimetics were unable to enhance the stimulatory effect of HDL. Blockade of PLC had no influence on progesterone secretion from cells treated with either NA or LH, but this did impair the stimulatory effect of insulin (P < 0.05). Similarly, blockade of PKC by staurosporine impaired (P < 0.05) the effect of insulin only but not that observed after LH or NA treatment. We suggest that: (a) noradrenergic stimulation does not enhance utilisation of cholesterol from HDL for progesterone secretion; (b) the fasting of luteal cells seems to activate enzymes responsible for the progesterone synthesis; (c) effect of NA on progesterone secretion from luteal cells does not involve the PLC-PKC pathway.     

Effect of constant infusion of oxytocin on luteal lifespan and oxytocin-induced release of prostaglandin F2α in heifers

Two experiments were conducted to determine whether constant infusion of oxytocin would prolong the luteal phase and inhibit uterine prostaglandin F2 alpha (PGF2 alpha) secretion in heifers. In Experiment 1, twelve heifers, treated with saline (SAL) or oxytocin (OXY) via jugular cannulae infusions (INF) or osmotic minipumps (OMP), were allotted at estrus into four treatment groups (n = 3). Treatments were: SAL-INF, SAL-OMP, OXY-INF and OXY-OMP. Physiological saline or oxytocin was given from Days 10 to 23 (Day 0 = estrus) of the estrous cycle. Method of treatment (jugular cannula infusion or osmotic minipump) had no effect (P greater than 0.05) on estrous cycle length or pattern of secretion of progesterone; therefore, data were pooled. Estrous cycle lengths were extended (P less than 0.01) for heifers which received oxytocin (25.3 +/- 0.4 d) compared to saline (20.5 +/- 0.4 d). Luteolysis did not occur in oxytocin-treated heifers until after treatment ceased. Experiment 2 was designed and conducted identically to Experiment 1 with the addition of a "challenge" injection of oxytocin (100 IU oxytocin, i.v.) given on Day 16 of the estrous cycle. Treatment of heifers with oxytocin extended (P less than 0.05) estrous cycle length by an average of 3 d compared to heifers treated with saline. The "challenge" injection induced (P less than 0.05) secretion of PGF2 alpha (as measured by the stable PGF2 alpha metabolite, 15-keto-13,14-dihydro-PGF2 alpha) in saline-treated but not oxytocin-treated heifers. In both Experiment 1 and 2, serum concentrations of FSH were elevated (P less than 0.05) in oxytocin-treated heifers. No increase was observed for LH or prolactin. The rise in estradiol-17 beta at luteolysis was not affected (P greater than 0.10) by treatment. In summary, constant infusion of oxytocin extended luteal lifespan, prolonged secretion of progesterone, and inhibited oxytocin-induced secretion of PGF2 alpha. Constant infusion of oxytocin did not affect serum concentrations of estradiol-17 beta, LH or prolactin; however, serum concentrations of FSH were elevated during the oxytocin treatment period.     

Changes in bovine luteal progesterone metabolism in response to exogenous prostaglandin F(2alpha)

An experiment was conducted to determine the effect of prostaglandin F2alpha (PGF2alpha) on luteal synthesis of progesterone (P4) and related progestins. Sixteen beef heifers were assigned in equal numbers to four groups in a 2x2 factorial arrangement of treatments. The experiment consisted of two levels of PGF2alpha analog (0 and 500 microg) and two levels of time (4 and 24 h after injection) of corpus luteum collection. All heifers were injected intravenously with saline (2 ml) or PGF2alpha (cloprostenol) on day 8 of the estrous cycle (estrus=day 0). Jugular blood was collected at 0, 1, 2, 3, 4 and 20, 21, 22, 23, and 24 h after injection. Resulting sera were analyzed for P4 by use of radioimmunoassay. Luteal tissue was analyzed by gas chromatography/mass spectrometry for P4, 20beta-hydroxyprogesterone, pregnenolone, and allopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one). Treatment with PGF2alpha reduced serum concentrations of P4 as early as 1 h after injection (P<0.005) and steroid levels remained low over 24 h. Similarly, administration of PGF2alpha caused a decline in luteal P4 (P<0.005), 20beta-hydroxyprogesterone (P<0.10), and pregnenolone (P<0.05). In contrast, treatment with PGF(2alpha) caused an increase in luteal allopregnanolone over time (time x treatment interaction; P<0.05). These data are interpreted to suggest that PGF2alpha promotes conversion of P4 to the metabolite allopregnanolone.     

Influence of progesterone, pregnenolone and 17beta-hydroxyprogesterone on the function of bovine luteal cells treated with luteinizing hormone, noradrenaline and prostaglandin E2

The aim of the present study was to investigate the influence of progesterone (P4), its precursor (pregnenolone; P5) and metabolite (17beta-hydroksyprogesterone; 17betaOHP4) on secretory function of bovine luteal cells on days 6-10 of the estrous cycle and on intracellular Ca2+ mobilization. The luteal cells were pre-incubated for 24 h and after change of medium they were incubated for 30 min with P5 and 17betaOHP4 (10(-5) each). Next, the medium was supplemented with LH (100 ng/ml), noradrenaline (NA; 10(-5) M) and prostaglandin (PG)E2 (10(-6) M), the cells were incubated for further 4 h and the medium was collected for P4 determination. Another set of luteal cells (5x10(4)/well) was incubated with P4, P5 and 17betaOHP4 at the dose of 10(-5) M each for 30 min and intracellular Ca2+ mobilization was measured every 5 s three times before and for 60 s after cells stimulation with LH, NA and PGE2. Metabolite of P4 did not affect the stimulatory effect of LH, PGE2 and NA on P4 secretion to the medium. Whereas all used steroids reduced calcium release from small but not from large luteal cells. It is suggested that steroids could temporary impair effect of luteotropins on the luteal cells via non-genomic way.     

The effect of progesterone on oxytocin-stimulated intracellular Ca2+ mobilisation and prostaglandin secretion in porcine endometrium

We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.     

Endocrine and cellular characteristics of corpora lutea from cows with a delayed post-ovulatory progesterone rise

The timing of the post-ovulatory progesterone rise is critical to the embryonic development and survival. The aim of this study was to determine the underlying causes of delayed post-ovulatory progesterone rises. Two groups of non-lactating dairy cows with early (n = 11) or late (n = 9) post-ovulatory progesterone rises were created by inducing luteolysis in the presence of either a large (> 10 mm) or small (< 10 mm) follicle, respectively. LH pulses were measured on days 4 (all cows) and 7 (n = 7, early; n = 5, late) (day 1= ovulation). The cows were slaughtered on day 5 (n = 4 each group) or 8 (n = 7, early; n = 5, late). Immunohistochemical analysis for endothelial cells (von Willebrand Factor, VWF), steroidogenic cells (3beta-HSD) and proliferation marker (Ki67) were performed. The basal progesterone production and LH responsiveness (0.001-100 ng/ml) of dispersed luteal cells was investigated. The luteal concentrations of FGF-2 and VEGF were measured by ELISA and RIA, respectively. There were no differences in LH pulse characteristics, area of VWF staining, proliferation index, steroidogenic cell characteristics, basal or LH-stimulated progesterone production by luteal cells between cows with an early or late progesterone rise (P > 0.10). However, the area of VWF staining increased from days 5 to 8, while the proliferation index decreased (P < 0.05). Furthermore, the luteal cells were more responsive to LH on day 8 (P < 0.01). Luteal concentrations of FGF-2 were higher on day 5 (P = 0.05), while VEGF was greater on day 8 (P < 0.01). In conclusion, we have clearly shown that LH support, degree of vascularization or luteal cell steroidogenic capacity were not the major factors responsible for inadequate secretion of progesterone by the developing bovine CL.     

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.     

Effect of an oxytocin antagonist on prostaglandin F2 alpha secretion and the course of luteolysis in sows.

The role of oxytocin (OT) in the regulation of prostaglandin F2 alpha (PGF2 alpha) secretion during luteolysis in gilts was studied using a highly specific OT antagonist (CAP-581). In Experiment 1 gilts on Days 14 to 19 of the oestrous cycle in Latin square design were used, to determine the dose and time of application of OT and CAP. In Group I (n = 6) gilts were treated intravenously with saline or with 10, 20 and 30 IU of OT. Concentrations of the main PGF2 alpha metabolite i.e. 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were measured in blood samples as uterine response to the treatment. Twenty IU of OT was the most effective to stimulate PGFM release and this dose was used after CAP treatment in gilts of Groups II, III and IV. Gilts of Group II (n = 3) were injected into the uterine horns (UH) with saline (5 ml/horn) or CAP (2 mg, 3 mg and 4 mg; half dose/horn) and OT was injected (i.v.) 30 min thereafter. Any of the CAP doses given into the UH affected PGFM plasma concentrations stimulated by OT. In Group III (n = 4) gilts were infused (i.v.) for 30 min with CAP (9 mg, 14 mg and 18 mg/gilt) followed by 20 IU of OT. All doses of CAP effectively inhibited OT-stimulated PGF2 alpha release, therefore 9 mg was selected for the further studies. Gilts of Group IV (n = 4) received OT 4, 6 and 8 h after CAP to define how long CAP blocks the OT receptors. Concentrations of PGFM increased after any of this period of time. Thus, we concluded that 9 mg of CAP infused every 4 h will effectively block OT receptors. In Experiment 2, gilts (n = 4) received CAP as a 30-min infusion every 4 h on Days 12-20 of the oestrous cycle. Control gilts (n = 3) were infused with saline. CAP infusions diminished the height of PGFM peaks (P < 0.05). Frequency of the PGFM (P < 0.057) and OT (P < 0.082) peaks only tended to be lower in the CAP-treated gilts. Peripheral plasma concentrations of progesterone (P4) and oestradiol-17 beta (E2) and the time of luteolysis initiation as measured by the decrease of P4 concentration were the same in CAP- and saline-treated gilts. The macroscopic studies of the ovaries in gilts revealed lack of differences between groups. We conclude that OT is involved in the secretion of luteolytic PGF2 alpha peaks but its role is limited to controlling their height and frequency. Blocking of OT receptors did not prevent luteolysis in sows.     

Immunohistochemical localization of steroidogenic enzymes in corpus luteum of wild sika deer during early mating season

We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 beta HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.     

Follicular, hormonal, and pregnancy responses of early postpartum suckled beef cows to GnRH, norgestomet, and prostaglandin F2alpha.

ABSTRACT: Cycling (n = 16) and noncycling (n = 24), early postpartum, suckled beef cows of three breeds were assigned randomly to three treatments: 1) 100-microg injection of GnRH plus a 6-mg implant of norgestomet administered on d -7 before 25 mg of PGF2alpha and implant removal on d 0 (GnRH+NORG); 2) 100 microg of GnRH given on d -7 followed by 25 mg of PGF2alpha on d 0 (GnRH); or 3) 2 mL of saline plus a 6-mg implant of norgestomet administered on d -7 followed by 25 mg of PGF2, and implant removal on d 0 (NORG). All cows were given 100 microg of GnRH on d +2 (48 h after PGF2alpha). Blood sera collected daily from d -7 to d +4 were analyzed for progesterone and estradiol-17beta, and ovaries were monitored daily by transrectal ultrasonography to assess changes in ovarian structures. Luteal structures were induced in 75% of noncycling cows in both treatments after GnRH, resulting in elevated (P < .01) progesterone on d 0 for GnRH+NORG-treated cows. Concentrations of estradiol-17beta (P < .01) and LH (P < .05) were greater on d +2 after GnRH for cows previously receiving norgestomet implants. Pregnancy rates after one fixed-time AI at 16 h after GnRH (d +2) were greater (P < .05) in GnRH+NORG (71%) than in GnRH (31%) and NORG (15%) cows. Difference in pregnancy rate was due partly to normal luteal activity after AI in over 87% of GnRH+NORG cows and no incidence of short luteal phases. The GnRH+NORG treatment initially induced ovulation or turnover of the largest follicle, induction of a new follicular wave, followed later by increased concentrations of estradiol-17beta and progesterone. After PGF2alpha, greater GnRH-induced release of LH occurred in GnRH+NORG cows before ovulation, and pregnancy rates were greater after a fixed-time AI.     

Influence of polychlorinated biphenyls on LH-stimulated secretion of progestereone and oxytocin from bovine luteal cells

Polychlorinated biphenyls (PCBs) due to their lipophilic properties can be easily accumulated in animal and human body and elicit diverse effects causing impairment of reproductive processes. Since these compounds were not be able to affect directly the luteal steroidogenesis, the aim of the present study was to verify hypothesis that PCBs can impair the effect of LH on the secretory function of luteal cells. Bovine luteal cells from different stages of the oestrous cycle (days 1-5, 6-10, 11-15 and 16-18) were exposed for 72h to various congeners of PCBs (PCB 126, PCB 77 and PCB 153) at the doses of 1, 10 or 100 ng/ml, in the presence or absence of LH (100 ng/ml), to determine the possible effect of these compounds on progesterone (P4) and ovarian oxytocin (OT) secretion. Only PCB 77 on days 1-5 and 16-18 increased P4 secretion. All PCBs decreased LH-simulated secretion of P4 from luteal cells obtained from all days of luteal phase. Dioxin-like congener (PCB 126) inhibited (P<0.05) the most evidently LH effect on P4 secretion. All congeners, except the lower doses of PCB 126, increased (P<0.05) OT secretion. They can also increase LH-stimulated secretion of OT, but the effect was dependent on the congener used and on the phase of oestrous cycle. On days 1-5 and 10-15, PCB 126 diminished LH-stimulated effect on OT secretion from luteal cells. PCB 77 (mimickig both dioxin and estradiol effect) in the higher doses, amplified effect of LH-stimulated OT secretion, while on all other days it diminished LH influence. PCB 153, which has estrogen-like properties, amplified LH effect on OT secretion during all studied days of the cycle. We conclude that PCBs (supposedly via estrogen and arylhydrocarbon - AhR receptor) may directly affect LH-stimulated function of CL. This does not appear to be a direct adverse effect on luteal steroidogenesis, but rather indirect on OT secretion from or within CL.