Low plasma progesterone concentrations are accompanied by reduced luteal blood flow and increased size of the dominant follicle in dairy cows

To investigate the influence of low plasma progesterone (P₄) concentrations on luteal and ovarian follicular development as well as endometrial gene expression in the concomitant and subsequent estrous cycle, 20 lactating dairy (Holstein Friesian and Brown Swiss x Holstein Friesian) cows received either a single treatment with 25 mg prostaglandin F_2α (PGF_2α) on Day 4 Hour 12 (PG1; n = 8), or two treatments (25 mg PGF_2α each) on Day 4 Hours 0 and 12 (PG2; n = 12) of the estrous cycle (Day 1, Hour 0 = ovulation). In four cows, ovulation occurred between 4 and 6 d after the second PGF_2α treatment; these cows and one lame cow were excluded. In the 15 remaining cows with physiological interovulatory intervals (18 to 24 d), P₄, luteal size (LS) and blood flow (LBF), as well as follicular size (FS) and blood flow (FBF), were determined daily until Day 4, immediately prior to (0 h) and 12 h after each PGF_2α treatment, and then every 2 d, from Day 5 to 8 d after the subsequent ovulation. Because P₄ did not differ (P > 0.05) between PG1 and PG2, cows were regrouped according to their mean P₄ concentration from Days 7 to 15, either P₄ <2 ng/mL (P₄L; n = 7) or P₄ >2 ng/mL (P₄H; n = 8). In the treatment cycle, LS was smaller in P₄L than P₄H on Days 13 (P = 0.01) and 15 (P = 0.03), and LBF was lower in P₄L than P₄H on Day 15 (P = 0.02). The dominant follicle of the first follicular wave was larger in P₄L than P₄H on Days 13 (P = 0.03), 15 (P = 0.03), and 17 (P = 0.01). In the subsequent cycle, there were no significant differences between P₄L and P₄H for P₄, FS, LS, and LBF; however, FBF was lower (P = 0.01) in P₄L than P₄H on Day 7. In Group P₄L, endometrial expressions of estrogen receptor α and oxytocin receptor were lower (P = 0.05 and P = 0.03, respectively) at the estrus that preceded treatment compared to the post-treatment estrus. In summary, low P₄ during diestrus was associated with smaller LS, reduced LBF, and larger FS in the treatment cycle, but not in the subsequent cycle.     

Effects of low versus physiologic plasma progesterone concentrations on ovarian follicular development and fertility in beef cattle

The objective of this study was to determine the effects of low versus physiologic plasma progesterone concentrations during the ovulatory wave on fertility in cattle. Suckled beef cows (Bos taurus; n = 129) and pubertal heifers (Bos taurus; n = 150) at random stages of the estrous cycle were given a luteolytic dose of prostaglandin F_2α (500 μg cloprostenol; PGF) twice, 11 d apart. Ten days after the second PGF treatment, cattle were given estradiol benzoate im (1.5 and 1.0 mg for cows and heifers, respectively) and a progesterone-releasing intravaginal device (Cue-Mate) with a single pod containing 0.78 g progesterone (Day 0). Cattle in the low-progesterone group (n = 148) received a luteolytic dose of PGF on Day 0, whereas those in the high-progesterone (i.e., physiologic plasma concentrations) group (n = 131) were allowed to retain their corpora lutea. On Day 8, the Cue-Mate was removed, and PGF was given to both groups. Fifty-four hours to 56 h later, cattle received 12.5 mg of porcine LH (pLH) im and were concurrently artificially inseminated. The dominant follicle in the low-progesterone group was larger (P < 0.001) than that in the high-progesterone group on the day of insemination (14.9 ± 0.3 mm vs. 12.7 ± 0.3 mm, mean ± SEM). At 7 d after ovulation, the low-progesterone group had a larger corpus luteum (24.5 ± 0.54 mm vs. 21.9 ± 0.64 mm, P < 0.01) and higher plasma progesterone concentration (4.0 ± 0.3 vs. 3.1 ± 0.2, P < 0.01) than that of the high-progesterone group. However, pregnancy rates did not differ (79 of 148, 53.4%, and 70 of 131, 53.4%) for low- and high-progesterone groups, respectively). In summary, low circulating progesterone concentrations during the growing phase of the ovulatory follicle resulted in a larger dominant follicle and a larger CL that produced more progesterone, with no significant effect on pregnancy rate.     

Evaluation of bovine luteal blood flow by using color Doppler ultrasonography

Since luteal vascularization plays a decisive role for the function of the corpus luteum (CL), the investigation of luteal blood flow (LBF) might give valuable information about the physiology and patho-physiology of the CL. To quantify LBF, usually Power mode color Doppler ultrasonography is used. This method detects the number of red blood cells moving through the vessels and shows them as color pixels on the B-mode image of the CL. The area of color pixels is measured with computer-assisted image analysis software and is used as a semiquantitative parameter for the assessment of LBF. Although Power mode is superior for the evaluation of LBF compared to conventional color Doppler ultrasonography, which detects the velocity of blood cells, it is still not sufficiently sensitive to detect the blood flow in the small vessels in the center of the bovine CL. Therefore, blood flow can only be measured in the bigger luteal vessels in the outer edge of the CL. Color Doppler ultrasonographic studies of the bovine estrous cycle have shown that plasma progesterone (P4) concentration can be more reliably predicted by LBF than by luteal size (LS), especially during the CL regression. During the midluteal phase, cows with low P4 level showed smaller CL, but LBF, related to LS, did not differ between cows with low and high P4 levels. In contrast to non-pregnant cows, a significant rise in LBF was observed three weeks after insemination in pregnant cows. However, LBF was not useful for an early pregnancy diagnosis due to high LBF variation among cows. When the effects of an acute systemic inflammation and exogenous hormones on the CL are examined, the LBF determination is more sensitive than LS assessment. In conclusion, color Doppler ultrasonography of the bovine CL provides additional information on luteal function compared to measurements of LS and plasma P4, but its value as a parameter concerning assessment of fertility in cows has to be clarified.     

Factors affecting spontaneous reduction of corpora lutea and twin embryos during the late embryonic/early fetal period in multiple-ovulating dairy cows

Spontaneous reduction of advanced twin embryos has been described in high-producing, Holstein-Fresian (Bos taurus) dairy herds. The first objective of the current study was to determine whether management and cow factors could have an effect on such a reduction in twin pregnancies during the early fetal period. Because loss of a corpus luteum was noted in cows suffering twin reduction, we expanded our study to include multiple-ovulating cows carrying singletons. Pregnancy was diagnosed and confirmed from Days 28 to 34 and 56 to 62 postinsemination. Sixty-nine (23.5%) of 293 pregnant cows with two corpora lutea carrying singletons and 132 (28.4%) of 464 twin pregnancies recorded on first pregnancy diagnosis subsequently lost one of the corpora lutea or one of the embryos, respectively. Thirty-four (25.8%) of the 132 twin pregnancies suffering embryo reduction lost one corpus luteum along with the embryo. Corpus luteum reduction always occurred in the ovary ipsilateral to the gravid horn suffering embryo reduction. Binary logistic regressions were performed considering corpus luteum and embryo reduction as dependent variables in single and twin pregnancies, respectively, and several management- and cow-related factors as independent variables. In cows carrying singletons, the risk of corpus luteum reduction was 14.3 (1/0.07) times lower for a given herd, whereas the interaction season by laterality significantly affected corpus luteum reduction such that in cows with two corpora lutea ipsilateral to the horn of pregnancy, the risk of reduction decreased during the winter period. In cows carrying twins, ipsilateral twin pregnancies were 3.45 (1/0.29) times more likely to undergo the loss of one embryo than bilateral twin pregnancies. As an overall conclusion, both corpora lutea and embryos were vulnerable to the effects of stress factors during the early fetal period in cows maintaining their pregnancies. A strong unilateral relationship between the corpus luteum and the conceptus was also observed.     

Effects of human chorionic gonadotropin on luteal blood flow and progesterone secretion in cows and in vitro-microdialyzed corpora lutea

To check human chorionic gonadotropin (hCG) effects on luteal blood flow (LBF) and progesterone (P₄) synthesis, six cows received either 3000 IU hCG or saline (NaCl) on Day 7 (Day 1 = ovulation) during two estrous cycles. Plasma P₄ and LBF were measured before (0 h) and up to 48 h after treatment. Luteal blood flow increased by 51% (P < 0.05) at 1 h after hCG administration and returned to baseline levels thereafter. Plasma P₄ levels were increased from pretreatment levels by 30% at 1 h (P = 0.05) and 81% at 48 h (P = 0.02) after hCG treatment. In contrast, NaCl did not cause changes in LBF and P₄ (P > 0.05). Additionally, central and peripheral parts of 14 abattoir-derived corpora lutea of the mid-luteal phase (Day 8 to 12) were perfused with Ringer solution in an in vitro microdialysis system, supplemented with 50 or 150 IU/mL hCG for 1 h. Application of 50 IU/mL hCG showed no influence on P₄ response (P > 0.05) in both central and peripheral parts, whereas 150 IU/mL hCG resulted in an increase of P₄ synthesis (P = 0.002) in the central parts only. In vivo, hCG provoked an immediate and long-term rise in P₄ but only a temporary elevation of LBF. Luteal blood flow itself does not seem to be the exclusive cause for an increase in P₄, because the in vitro data clearly showed direct effects of hCG on P₄ secretion. Interestingly, different P₄ secretion patterns could be found between central and peripheral parts of the corpus luteum in both control and hCG perfused corpora lutea.     

Luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size

The objective of this study was to assess the reliability of luteal blood flow (LBF) as recorded by color Doppler sonography to monitor luteal function during the estrous cycle of dairy cows and to compare the results with that for the established criterion luteal size (LS) as determined by B-mode sonography. In total, 14 consecutive sonographic examinations were carried out in 10 synchronized lactating Holstein-Friesian cows (Bos taurus) on Days 4, 5, 6, 7, 8, 10, 12, 14, 16, -5, -4, -3, -2, -1 of the estrous cycle (Day 1 = ovulation). Plasma progesterone concentrations in venous blood (P₄) were quantified by enzyme immunoassay. Luteal size was determined by sonographic measurement of the maximal cross-sectional area of the corpus luteum (CL). Luteal blood supply was estimated by calculating the maximum colored area of the CL from power Doppler sonographic images. Luteal size doubled during the luteal growth phase (until Day 7) and remained at this level during the luteal static phase (Day 8 to 16) before decreasing rather slowly during luteal regression (Days -5 to -1). Luteal blood flow doubled during the growth phase, doubled furthermore during the static phase, and decreased rapidly during luteal regression. Thus, LBF values represented highly reliable predictors of luteal status. Luteal blood flow predicted reliably a P₄ > 1.0 ng/mL by reaching only 35% of the maximal values, whereas LS had to exceed 60% of the maximal values to indicate reliably a functional CL. It is concluded that LBF reflected luteal function better than LS specifically during luteal regression.     

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D−10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N = 42) or not (small follicle-small CL group; SF-SCL; N = 41) on D−10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 ± 0.33 mm vs. 10.76 ± 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 ± 0.28 pg/mL vs. 1.27 ± 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 ± 0.25 ng/mL vs. 2.62 ± 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.     

Relation between progesterone concentrations during the early luteal phase and follicular dynamics in goats

We studied the relationship between progesterone (P4) concentrations early in the estrus cycle and follicular dynamics in dairy goats. We used seven untreated goats (control group) and six progesterone treated goats (P group) with a controlled internal drug release device from Days 0 to 5 (Day 0: day of ovulation). We performed daily ultrasonograph during the interovulatory interval to determine ovarian change and took daily blood samples to determine serum estradiol 17beta (E2) and P4 concentrations by RIA. We divided the control goats into 3- (n = 4) and 4-wave goats (n = 3), according to the number of follicular waves recorded during the ovulatory cycle. Mean progesterone concentrations between Days I and 5 were higher and mean estradiol concentrations between Days 3 and 5 were lower in 4-wave goats (P4: 3.8+/-0.2 ng/ml; E2: 1.6+/-0.2 pg/ml) than in 3-wave goats (P4: 2.0+/-0.5 ng/ml, P < 0.05; E2: 4.4+/-0.9 pg/ml, P < 0.05). Wave 2 emerged earlier in 4-wave (Day 4.2+/-0.3) than in 3-wave goats (Day 7.3+/-0.3, P < 0.05). Three out of six of the progesterone-treated goats had short cycles (mean 8.0+/-0.0 days) and ovulated from Wave 1. The other three goats had shorter cycles (mean 18.3+/-0.3 days) than the control group (20.0+/-0.2 days; P < 0.05), although they were within the normal range of control cycles (shortened cycles). In the three treated goats with shortened cycles (two with four waves, one with three waves), mean progesterone concentrations between Days I and 5 were higher (4.7+/-0.6 ng/ml) than in the 3-wave control goats. In these goats, Wave 2 emerged at Day 4.3+/-0.3, similar to the time observed in 4-wave goats but earlier (P < or = 0.05) than in 3-wave control goats. Overall results confirm a relationship between the progesterone levels and the follicular wave turnover during the early luteal phase in the goat. Higher progesterone concentrations may accelerate follicular turnover probably by an early decline of the negative feedback action of the largest follicle of Wave 1. This is followed by an early emergence of Wave 2.     

Nuclear volume and chromatin conformation of small and large bovine luteal cells: effect of gonadotropins and prostaglandins and dependence on luteal phase

Summary Change in nuclear volume and chromatin conformation are generally considered to reflect altered gene expression in eukaryotic cells. The present studies were undertaken to investigate whether these nuclear parameters of luteal cells can be altered by hormone treatment in vitro or change during the estrous cycle. The nuclear volume of small luteal cells was significantly lower than that of large luteal cells during the cycle and pregnancy. The nuclear volumes of small and large luteal cells from pregnancy did not change during incubation without any hormone or with 10 nM prostaglandin (PG)F₂. However, incubation with 1 nM human chorionic gonadotropin (hCG) or 10 nM PGE₁ resulted in a significant increase of nuclear volume of small luteal cells by 4 h and that of large luteal cells by 6 h. Small cells were more responsive to hCG than large luteal cells. The nuclear volumes of small and large luteal cells also significantly increased from early to mid luteal phase with no further change in late luteal phase. hCG and PGE₁, as well as PGF₂, treatment resulted in a change of chromatin conformation of small and large luteal cells. Dibutyryl cyclic AMP (10 mM) mimicked the hormones by increasing nuclear volumes and changing the chromatin conformation of small and large luteal cells. Chromatin conformation of small and large luteal cells also changed from early to mid luteal phase and mid to late luteal phase. In conclusion, in vitro, hCG and PGs can regulate nuclear volume and/or chromatin conformation of small as well as large bovine luteal cells. In vivo, these nuclear changes occur during the periods of luteal growth, development and regression in the estrous cycle.     

Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch

In nonpregnant and pregnant dogs the corpora lutea (CL) are the only source of progesterone (P4) which shows an almost identical secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained that physiological luteal regression is devoid of a functional role of the PGF2α-system and seems to depend on the provision of StAR. Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2α, may be indicative for different regulatory mechanisms. To assess this situation and by applying semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant and prepartal dogs and of mid-pregnant dogs treated with the antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin E2 synthase (PGES), prostaglandin F2α synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid-dehydrogenase (3βHSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were determined by RIA. CL were collected via ovariohysterectomy from pregnant bitches (n = 3–5) on days 8–12 (Group 1, pre-implantation period), days 18–25 (Group 2, post-implantation period), days 35–40 (Group 3, mid-gestation period) and during the prepartal progesterone decline (Group 4). Additionally, CL were obtained from groups of 5 mid-pregnant dogs (days 40–45) 24 h, respectively 72 h after the second treatment with Aglepristone. Expression of Cox2 and PGES was highest during the pre-implantation period, that of PGFS and FP during the post-implantation period. EP4 and EP2 revealed a constant expression pattern throughout pregnancy with a prepartal upregulation of EP2. 3βHSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it was matched by the course of P4 concentrations. Expression of the PR was higher during mid-gestation and prepartal luteolysis than in the two preceding periods. After application of Aglepristone the overall mRNA-expression resembled the situation during prepartal luteolysis except for EP2, which remained unchanged.These data suggest that – as in the nonpregnant bitch – also in the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand induction of luteolysis by the PR blocker Aglepristone points to a role of luteal P4 as an autocrine factor in a positive loop feedback system controlling the availability of P4, StAR and 3βHSD.     

Alterations in luteal production of androstenedione,testosterone, and estrone,but not estradiol,during mid- and late pregnancy in pigs: Effects of androgen deficiency

Recently, we have found that flutamide-induced androgen deficiency altered progesterone production in the porcine corpus luteum (CL) during mid- and late pregnancy. Herein, we tested whether flutamide administration subsequently influences androgen and estrogen metabolism in the CL of pregnancy. Pregnant gilts were treated with flutamide between Days 43 and 49 (GD50F), 83 and 89 (GD90F), or 101 and 107 (GD108F) of gestation. Corpora lutea (CLs) were collected from treated and nontreated (control) pigs. The concentrations of androstenedione (A4), testosterone (T), estrone (E1), and estradiol (E2) together with the levels of expression of mRNAs and proteins for cytochrome P450 17α-hydroxylase/c17-20 lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), cytochrome P450 aromatase (CYP19A1), and 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) were measured in the CL of control and flutamide-treated animals. Steroidogenic enzymes were also immunolocalized in luteal tissues. The luteal concentrations of A4 and T were higher in the GD50F (P = 0.006, P = 0.03) and GD108F (P = 0.005, P = 0.035) groups, but lower in the GD90F (P = 0.004, P = 0.014) group. The E1 level was greater only in the GD90F (P = 0.03) and GD108F (P = 0.035) groups, whereas E2 concentration was not affected by flutamide treatment. Increased luteal CYP17A1 mRNA and protein expression was found in the GD50F (P = 0.002, P = 0.03) and GD108F (P = 0.0026, P = 0.03) groups, but reduced in the GD90F (P = 0.002, P = 0.03) group. mRNA of 17β-HSD1 was upregulated in the GD50F (P = 0.0005) group, but downregulated in the GD90F (P = 0.002) and GD108F (P = 0.0005) groups. In contrast, 17β-HSD1 protein expression was higher in the GD50F and GD108F (P = 0.03) groups, but lower in the GD90F (P = 0.03) group. Both CYP19A1 mRNA and protein levels were greater in the GD90F (P = 0.001, P = 0.028) and GD108F (P = 0.005, P = 0.03) groups. Neither 17β-HSD7 mRNA nor protein level were affected by flutamide exposure. Both CYP17A1 and 17β-HSD1 were immunolocalized exclusively in small luteal cells, whereas CYP19A1 and 17β-HSD7 were found in large luteal cells of control and flutamide-treated CLs. Overall, flutamide administration led to the alterations in A4, T, and E1, but not in E2, production in the CL of pregnancy in pigs, probably because of disrupted steroidogenic enzymes expression. These changes suggest that androgens are important modulators of luteal function during pregnancy in pigs.     

Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin-like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8-Br-cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na(+)-K(+)-ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells.     

Corpus luteum development and function and relationship to pregnancy during the breeding season in the Mediterranean buffalo

The aim of this study was to ascertain corpus luteum (CL) development and function in buffaloes synchronized and mated by artificial insemination (AI) during the breeding season. Italian Mediterranean buffalo cows (n = 43) at 86.5 ± 2.7 days postpartum were synchronized by the Ovsynch-TAI Program and inseminated using frozen thawed semen at 20 and 44 h after the second injection of GnRH. The CL dimensions (diameter and area) and blood flow were examined on Days 5, 10, 15, 20, and 25 after AI by realtime B-mode/colour-Doppler ultrasonography. The resistive index (RI), pulsatility index (PI) and time average medium velocity (TAMV) were recorded at each time, together with CL dimensions. Blood samples were taken on the days of ultrasonography for progesterone (P4) assay by RIA. Data were grouped into pregnant or non-pregnant and retrospectively analyzed by repeated measure ANOVA and correlation analyses. Dimensions of the CL on Days 10, 20, and 25 after AI were greater (P < 0.01) in buffaloes pregnant on Day 45 (n = 18) compared with non-pregnant buffaloes (n = 25). The former buffaloes also showed a greater (P < 0.01) rate of CL growth between Days 5 and 10 after AI. Blood flow to the CL on Day 10 after AI showed a higher TAMV (P < 0.01) and lower RI (P < 0.05) in pregnant buffaloes compared with non-pregnant buffaloes. Negative correlations were observed on Day 10 after AI between CL diameter and RI (r = −0.61; P < 0.01) and PI (r = −0.60; P < 0.01); P4 concentrations and RI (r = −0.46; P < 0.02); and RI and pregnancy (r = 0.45; P < 0.02). Positive correlations were observed between pregnancy and CL size (r = 0.54; P < 0.01), ΔCL diameter between Days 5 and 10 (r = 0.52; P < 0.01), ΔCL area between Days 5 and 10 (r = 0.48; P < 0.015), and ΔP4 between Days 5 and 10 (r = 0.50; P < 0.01). Based on these findings it is concluded that the period between Day 5 and 10 is very important for CL growth and crucial in evaluating pregnancy. Accordingly, the assessment of CL parameters during the period from Day 5 to Day 10 after AI might be used to predict the likelihood of an ongoing pregnancy.     

Effect of high progesterone concentrations during the early luteal phase on the length of the ovulatory cycle of goats

The effect of exogenous progesterone exposure early in the oestrous cycle on the duration of the interovulatory interval was studied in dairy goats. A controlled intravaginal drug release (CIDR-G) device was inserted for 5 days starting at day 0 (D0 group, n=6) or day 3 (D3 group, n=5) postovulation. A third group was composed of untreated control goats (control group, n=7). Daily transrectal ultrasound was carried out during the interovulatory interval to assess the ovarian dynamics. Oestrous behaviour was checked twice a day and serum progesterone levels were assayed in daily jugular blood samples. Treated goats showed two different responses. In three D0 goats and one D3 goat, progesterone concentrations fell immediately after CIDR withdrawal and this was followed by oestrus and ovulation between days 8 and 11 (short cycles). In the other three D0 goats and in four D3 goats the treatment significantly reduced the interovulatory interval (18.3+/-0.3 and 18.5+/-0.3 days, respectively) (shortened cycles) compared with the control group (20.0+/-0.2 days; P<0.05), but the intervals with progesterone concentrations over 1 ng/ml were not different (15.7+/-0.3, 15.8+/-0.7 and 16.0+/-0.5 days for D0, D3 and control goats, respectively). In all D0 goats with a short cycle response, the ovulatory follicle arose from the first follicular wave but in the D3 goat with a short cycle it arose from the second follicular wave. These results showed that premature progesterone exposure early in the ovulatory cycle of the goat affected its length inducing short or shortened cycles. The effect of progesterone could either affect luteotropic support of the corpus luteum (CL) and/or stimulate a premature release of the luteolysin.     

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: Tissue culture approach

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P₄) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.     

Luteal inadequacy during the early luteal phase of subfertile cows

A study was made of early luteal function (up to Day 6) in cyclic and pregnant heifers and also in older, subfertile cows. There were no differences in vivo or in vitro between cyclic and pregnant heifers, indicating no luteotrophic effect of the embryo at this stage, but the increase in postovulatory peripheral progesterone concentrations was delayed (P less than 0.01) and occurred more slowly (P less than 0.001) in the subfertile cows than in the heifers. The corpora lutea of the subfertile cows were heavier (P less than 0.001) than those of the heifers on Day 6. Basal progesterone production by dispersed luteal cells was similar between heifers and subfertile cows, but there was a difference (P less than 0.001) in the pattern of response to exogenous LH and PGE-2. Cells from subfertile cows were less sensitive to the stimulatory effects of PGE-2 and although LH increased (P less than 0.001) progesterone production by all cells, this stimulation by a low dose of LH was inhibited by PGE-2 in luteal cells from subfertile cows. This effect did not occur in the luteal cells from heifers. These results indicate the possibility that luteal inadequacy, due to a diminished response to circulating luteotrophic hormones, may contribute to embryo mortality in subfertile cows.     

Dominant expression of porcine Calbindin-D9k in the uterus during a luteal phase

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue.     

Influence of oxytocin infusion during oestrus and the early luteal phase on progesterone secretion and the establishment of pregnancy in ewes

In Experiment 1, an osmotic minipump containing oxytocin was implanted s.c. in ewes for 12 days beginning on Day 10 of the oestrous cycle, producing approximately 100 pg oxytocin/ml in the plasma. Two days after the start of infusion, all ewes were injected with 100 micrograms cloprostenol and placed with a fertile ram. At slaughter 22 days later, 9 (75%) of the 12 control (saline-infused) ewes were pregnant compared with 1 (11%) of the 9 ewes infused with oxytocin. In the control group, midcycle plasma concentrations of oxytocin were significantly higher in nonpregnant than in pregnant ewes. In Experiment 2, an infertile ram was used throughout to avoid any possible effects of pregnancy and oxytocin infusions were given at different stages of the oestrous cycle. Otherwise the protocol was similar to that in Exp. 1. Oxytocin infusion during luteolysis and the early follicular phase had no effect on the subsequent progesterone secretion pattern, but infusions beginning the day before cloprostenol-induced luteolysis and lasting for 7 or 12 days and infusions beginning on the day of oestrus for 4 days all delayed the subsequent rise in plasma progesterone by approximately 3-4 days. In these animals, the cycle tended to be longer. It was concluded that an appropriate oxytocin secretion pattern may be necessary for the establishment of pregnancy in ewes and that a high circulating oxytocin concentration during the early luteal phase delays the development of the young corpus luteum.