Collagen and matrix metalloproteinase-2 and -9 in the ewe cervix during the estrous cycle.

The cervical collagen remodeling during the estrous cycle of the ewe was examined. The collagen concentration determined by a hydroxyproline assay and the area occupied by collagen fibers (%C), determined by van Gieson staining, were assessed in the cranial and caudal cervix of Corriedale ewes on Days 1 (n = 6), 6 (n = 5), or 13 (n = 6) after estrous detection (defined as Day 0). In addition, the gelatinase activity by in situ and SDS-PAGE gelatin zymographies and matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9, respectively) expression by immunohistochemistry were determined. The collagen concentration and %C were lowest on Day 1 of the estrous cycle (P < 0.04), when MMP-2 activity was highest (P < 0.006) and the ratio of activated to latent MMP-2 trend to be highest (P = 0.0819). The MMP-2 activity was detected in 73% of the homogenized cervical samples, and its expression was mainly detected in active fibroblasts. By contrast, the MMP-9 activity was detected in 9% of the samples, and its scarce expression was associated with plasmocytes, macrophages, and lymphocytes. Matrix metalloproteinase-2 expression was maximal on Day 1 in the cranial cervix and on Day 13 in the caudal cervix and was lower in the cranial than in the caudal cervix (P < 0.0001). This time-dependent increase in MMP-2 expression that differed between the cranial and caudal cervix may reflect their different physiological roles. The decrease in the collagen content and increase in fibroblast MMP-2 activity in sheep cervix on Day 1 of the estrous cycle suggests that cervical dilation at estrus is due to the occurrence of collagen fiber degradation modulated by changes in periovulatory hormone levels.

Cervical changes in estrogen receptor alpha, oxytocin receptor, LH receptor, and cyclooxygenase-2 depending on the histologic compartment, longitudinal axis, and day of the ovine estrous cycle.

The aim was to investigate the histologic distribution of estrogen receptor α (ERα), oxytocin receptor (OxR), LH receptor (LHR), and cyclooxygenase-2 (COX-2) in the cervix of the ewe during the estrous cycle. Immunohistochemistry was performed in the cranial and caudal cervix of Corriedale ewes on Day 1 (n = 6), 6 (n = 5), or 13 (n = 6) after estrous detection (Day 0). The ERα proportional score (%ERα nuclei) was lower in the cranial cervix than in the caudal cervix, whereas the OxR and COX-2 immunostaining areas (%areas) were greater in the cranial cervix than in the caudal cervix (P < 0.04). The %ERα nuclei decreased from Days 1 to 13 in luminal epithelia, but increased from Days 1 to 6 or remained unchanged in stromata (P < 0.003). The %OxR area was higher on Day 6 than on Days 1 and 13 in the superficial glandular epithelium, and increased from Days 1 to 13 in the deep glandular epithelium (P < 0.04). The %LHR area increased during the estrous cycle in luminal epithelia and fold stroma (P < 0.004). The %COX-2 area was restricted to epithelia, and it was lower on Day 1 than on Days 6 and 13 in luminal epithelia (P < 0.05). Differences in ERα, OxR, LHR, and COX-2 between cranial and caudal cervical zones indicate different physiological functions, and their cyclic variations in the cervical epithelia, in contrast to little or no variations in the stroma, suggest a hormone-responsive driving role of epithelia in cervical function.

Expression of genes for oestrogen and progesterone receptors in the cervix of anoestrous ewes treated with gonadotrophin releasing hormone with or without progesterone priming.

The aim was to determine the oestrogens receptor alpha (ERα) mRNA and the binding capacity of oestrogens (ER) and progesterone receptor (PR) in the cervix of anoestrous ewes treated with gonadotrophin-releasing hormone (GnRH) with or without progesterone (P) priming, at the expected time of induced ovulation and early luteal phase. In Experiment 1, ewes were treated with P for 10 days (n=4), with nine micro-doses of GnRH followed by a GnRH bolus injection (n=4), or with P plus GnRH treatments (n=3), and tissues were harvested either without treatment (n=4), when P was removed, or 24h after the GnRH bolus injection. In Experiment 2, ewes were treated with the same GnRH or P plus GnRH treatments and tissues were harvested on Day 1 (n=12) or Day 5 (n=10) after the GnRH bolus injection. In the cranial cervix, the P treatment decreased and the GnRH treatment (after P treatment) increased the ERα mRNA, ER and PR concentrations (P<0.002). The ERα mRNA and ER concentrations were greater on Day 1, than on Day 5 in P plus GnRH treated ewes (P<0.0005). In the caudal cervix, lesser ERα mRNA, ER and PR concentrations than cranial cervix were found (P<0.0001). In conclusion, the ERα transcriptional activity and ER and PR binding capacity were strongly influenced by P and/or GnRH treatments in the cranial cervix, while the steroid receptors binding capacity remained unchanged in the caudal cervix of anoestrous ewes at the expected time of induced ovulation and early luteal phase.

Oestrogen and progesterone receptor binding capacity and oestrogen receptor alpha expression (ERalpha mRNA) along the cervix of cycling ewes.

The aim of the present work was to study the oestrogen receptor (ER) and progesterone receptor (PR) binding capacity and the oestrogen receptor alpha (ERalpha) mRNA concentration in cranial and caudal cervix during the ovine oestrous cycle. Cervical samples of synchronised Corriedale ewes were obtained on Day 1 (n=7), 6 (n=6) or 13 (n=7) after oestrus detection (Day 0). The ER and PR binding capacity by ligand-binding assay and the ERalpha mRNA concentration by solution hybridisation in both cranial and caudal zones of the cervix were determined. The ER and PR binding capacity were higher (P<0.005) on Day 1 than on Days 6 and 13 in both cranial and caudal zones. The ERalpha mRNA concentrations were higher (P<0.0001) on Day 1 than on Days 6 and 13 only in the caudal zone. The PR binding capacity and ERalpha mRNA concentration were higher (P<0.005) in the caudal than in the cranial zone on Day 1. The ER and PR expression in the ovine cervix varied during the oestrous cycle in agreement with the known upregulation exerted by oestrogen and downregulation exerted by progesterone. Differences in ER and PR expression along the longitudinal axis of the ovine cervix were found, reflecting histological and functional differences between the cranial and caudal zones.

Effect of undernutrition on uterine progesterone and oestrogen receptors and on endocrine profiles during the ovine oestrous cycle.

In the present study, it was investigated whether undernutrition affected the binding capacity, immunoreactivity and mRNA expression for uterine oestrogen and progesterone receptors (ER and PR, respectively) in sheep, as well as whether the responses were associated with changes in plasma concentrations of progesterone (P4), oestradiol (E2), glucose, fatty acids, insulin, leptin and insulin-like growth factor (IGF)-I during the oestrous cycle. Twenty ewes were fed either 1.5 (C) or 0.5 (L) times their maintenance requirements and were killed on Day 5 or 14 of the cycle (Day 0 = oestrus). Compared with Group C, Group L had higher concentrations of non-esterified fatty acids and lower concentrations of insulin, leptin and IGF-I. Group L also had higher plasma concentrations of P4 during the final days of the luteal phase. At oestrus in both treatment groups, there were peaks in the concentrations of glucose, insulin and IGF-I. For ER and PR, transcript expression, binding capacity and immunoreactivity were higher on Day 5 than on Day 14 of the cycle. The binding capacities for ER and PR were lower in Group L than in Group C on Day 5. Group C showed more immunoreactive staining for ER than did Group L in two of five cell types, whereas no effect of treatment was observed for PR immunoreactivity. There was more PR mRNA in the uterine horn contralateral to the corpus luteum in Group C than in Group L ewes. We conclude that undernutrition impairs steroid receptor expression and binding capacity. This may alter the uterine environment and help explain the reductions in embryo survival.

Corpus luteum life span and pituitary oestrogen and progesterone receptors in cyclic and gonadotrophin-releasing hormone-treated anoestrous ewes.

The present study investigated the pituitary oestrogen (ER) and progesterone (PR) receptor concentrations in ewes during the oestrous cycle in the breeding season (n = 19), and in anoestrous ewes treated with gonadotrophin-releasing hormone (GnRH) (n = 11) and anoestrous ewes treated with progesterone + GnRH (n = 11). The pituitary ER and PR concentrations at the expected time of ovulation and in the early and late luteal phases were measured by binding assay. The pattern of pituitary ER and PR concentrations in the progesterone + GnRH-treated ewes resembled the pattern found during the normal oestrous cycle, with ER and PR concentrations decreasing from the time of ovulation to the early luteal phase. In contrast, in ewes treated with GnRH alone, ER and PR concentrations increased in the early luteal phase, which may increase the inhibitory effects of steroid hormones on luteinising hormone secretion, ultimately leading to the development of subnormal luteal phases.

Experimentally induced subnormal or normal luteal phases in sheep: reproductive hormone profiles and uterine sex steroid receptor expression.

This study investigated if ewes expected to have subnormal luteal phases (SNLP) present a different pattern of uterine oestrogen receptor (ER) and progesterone receptor (PR) expression at the expected time of premature luteolysis. The concentrations of uterine ER, PR and ERalpha mRNA, and the steroid ovarian hormone were determined in anoestrous ewes treated with either gonadotrophin-releasing hormone (GnRH) to develop a SNLP (n = 16), or progesterone + GnRH to develop a normal luteal phase (NLP; n = 16). The ER, PR and ERalpha mRNA concentrations were measured using binding and solution hybridisation assays, while the hormone level concentration was measured by radioimmunoassay. In all ewes, a luteinising hormone- and follicle-stimulating hormone-synchronised surge was found. The SNLP group had lower preovulatory oestradiol levels than the NLP group. On Day 5, the SNLP group had lower progesterone levels, and higher uterine ER, PR and ERalpha mRNA concentrations than the NLP group. While in the SNLP group the receptor expression increased from Days 1 to 5, in the NLP group the receptor expression decreased. The results suggest that the induction of steroid receptor expression in the uterus and the hormonal environment found in the experimental SNLP group at the expected time of premature luteolysis may be involved in the mechanisms causing SNLP.

Differential estradiol effects on estrogen and progesterone receptors expression in the oviduct and cervix of immature ewes.

In order to study the effect of estradiol-17beta (E2) on estrogen (E) and progesterone (P) receptors expression in oviduct and cervix of lambs, their respective transcripts (ERalpha mRNA and PR mRNA) were determined by solution hybridization and the receptor proteins (ER and PR) by binding assays after E2 treatments. Lambs (n=4 in each group) were not treated or treated with one, two or three i.m. injections of E2 (1 microg/kg) at 24 h of interval. Tissues were obtained 12 or 24 h after the last E2 injection. Estradiol treatments increased ERalpha mRNA and PR mRNA concentrations in an organ-dependent manner: transitory in the oviduct while maintained in the cervix. The E2 effect on the oviductal and cervical ER and PR concentrations were biphasic, with an initial reduction of receptors content that was followed by restoration. The ER restoration in oviduct was earlier than in the cervix. In summary, this study shows that E2 treatments may exert an inductive effect in ERalpha mRNA and PR mRNA levels and a biphasic effect in ER and PR concentrations in oviduct and cervix of immature ewe. These E2 effects varied in timing and strength depending on the organ of the reproductive tract.

The role of sex steroid receptors in sheep female reproductive physiology.

Cell responsiveness to steroid hormones is related to the number and affinity of its receptors, thus factors affecting steroid expression will influence tissue sensitivity and functionality. The present review discusses the role of oestrogen and progesterone receptors in sheep female reproductive physiology. The mechanism of steroid hormone action in the target cell is introduced first; the tissue distribution, physiological functions and regulation of oestrogen receptor subtypes and progesterone receptor isoforms in ruminants are reported. The role of steroid receptors in target tissues (with emphasis on the uterus and pituitary gland) during different physiological events is addressed in an attempt to clarify oestrogen and progesterone actions in different developmental and reproductive stages: prepubertal period, oestrous cycle, pregnancy, post-partum period and seasonal anoestrus. The present review shows how the distinct reproductive stages are accompanied by dramatic changes in uterine receptor expression. The role of oestrogen and progesterone receptors in the molecular mechanism responsible for premature luteolysis that results in subnormal luteal function is discussed. Finally, the effect of nutrition on sex steroid receptor expression and the involvement on reproductive performance is reported.

Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes.

The objective of this work was to investigate the effect of progesterone (P) and gonadotropin-releasing hormone (GnRH) treatment on estrogen receptor (ER) and P receptor (PR) concentrations in the pituitary gland and uterus of anestrous ewes. Ewes were either not treated (group C, n = 4); were treated with 0.33 g P-controlled internal drug release (P-CIDR) for 10 days (group P, n = 4), with GnRH, 6.7 ng i.v. injections every 2 h for 18 h followed by a 4 microg bolus administration of Receptal at 20 h (group GnRH, n = 4), or with a combination of the P and GnRH treatment (group P + GnRH, n = 3). Ewes were humanely killed either at the beginning of the experiment (group C), when the CIDR was removed (group P), or 24 h after the GnRH bolus treatment (groups GnRH and P + GnRH). Progesterone treatment increased serum P concentrations, indicating that the treatment was effective. All GnRH treated ewes had similar luteinizing hormone (LH) surges, which lasted 8 h. At slaughter, estradiol (E2) concentrations in the GnRH group were higher than in groups C, P, and P + GnRH. Treatment with GnRH increased more than 10-fold the content of ER and PR in the pituitary gland without altering steroid receptor concentrations in the uterus. When GnRH was combined with P the uterine receptor contents were higher than with P treatment alone. The treatment with P decreased ER and PR content in the uterus, but had no effect on the pituitary gland. The results show that regulation by P and GnRH of ER and PR content in anestrous ewes is tissue-specific.

Regulation by gonadal steroids of estrogen and progesterone receptors along the reproductive tract in female lambs.

The regulation of estrogen and progesterone receptor (ER, PR) expression by estradiol (E2) and progesterone (P4) in the oviduct, uterus and cervix of female lambs was studied. The animals received three intramuscular injections of E2, P4 or vehicle with an interval of 24 h and they were slaugthered 24 h after the third injection. Determinations of ER and PR were performed by binding assays and mRNAs of ER alpha and PR by solution hybridization. High levels of ER and PR in both cervix and oviduct were found in the female lamb, differing from other mammalian species. No significant effects by either E2 or P4 treatment on ER and PR levels in the cervix and oviduct could be observed. E2 treatment increased the mRNA levels of ERa and PR more than 3-fold in the cervix, while P4 treatment increased the mRNA levels of ERa and PR in the uterus. The results show differential effects of gonadal steroids on sex steroid receptor expression along the reproductive tract in female lambs, suggesting that steroid target tissues can modulate responses to the same circulating levels of steroid hormones.

Regulation of insulin-like growth factor-I and thioredoxin expression by estradiol in the reproductive tract of the prepubertal female lamb.

Estradiol (E(2)) has been shown to be an important uterine growth promoting molecule in the ovariectomized (ovx) rat, which increases the mRNA levels of insulin-like growth factor-I (IGF-I) and the redox enzyme thioredoxin. The aim of this study was to explore the role of E(2) in the regulation of IGF-I and thioredoxin in the reproductive tract of the prepubertal female lamb. Twenty 3-month-old lambs were treated with i.m. injections of E(2) at 24 h intervals. The animals were sacrificed 12 or 24 h after the last injection, and 72 h was the longest treatment period. The mRNA levels of thioredoxin and IGF-I were determined by a solution hybridization technique. There was a 5-fold increase in the cervical IGF-I mRNA level 12 h after the first E(2) injection. The uterine IGF-I mRNA level was doubled after 12 h and this increase was maintained during the rest of the experimental period. The IGF-I mRNA level in the oviducts was more than doubled 12 and 24 h after the E(2) injection, then the level decreased towards the initial level. The thioredoxin mRNA level in the cervix was increased 4-fold after 24 h, whereas no significant effect was seen in the uterus. The thioredoxin mRNA level in the oviduct was more than doubled 12 and 24 h after the first E(2) injection. Thus, estradiol regulates the expression of IGF-I and thioredoxin in the reproductive tract of prepubertal lambs.

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus.

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol- 17beta. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.

Estrogen and progesterone receptors in the ovine cervix during the postpartum period.

Cervical estrogen (E) and progesterone (P) receptors were characterized and quantified during the postpartum period in Corriedale ewes lambing in the late breeding season. Cervices and uteri were collected after ovariohysterectomy at 1 d (n = 2), 5 d (n = 4), 17 d (n = 2) or 30 d (n = 2) post partum. The estrogen and progesterone receptors were measured using binding assays with tritiated hormones, dextran charcoal separation and inverse Scatchard analysis. Similar kinetic parameters in cytosolic binding sites for both hormones were found in all cervical and uterine samples, indicating that the binding protein in both tissues is of the same nature. Receptor concentrations (fmol/mg cytosolic protein) in the cervix of early (1 to 5 d, n = 6) and late (17 to 30 d, n = 4) postpartum ewes were 348 +/- 66 vs 994 +/- 145 (P < 0.05) for E and 618 +/- 126 vs 1170 +/- 201 (P < 0.05) for P, respectively. These data suggest an increased synthesis of receptors, probably due to the presence of ovarian estrogen-active follicles. Cervical E and P receptor concentrations were similar or higher than those in the uterus (1.40 +/- 0.15, n = 10 and 1.51 +/- 0.19, n = 10; for E and P respectively), and these receptor ratios did not differ between the early and late postpartum period. The high ratio between cervical/uterine receptors suggests that the ovine cervix may be a very sensitive to steroid action. In conclusion, it was shown that restoration of steroid receptors during the postpartum period in the ovine cervix is similar to receptor dynamics in the uterus, and is probably associated with the recovery of ovarian cyclicity.

Priming effect of exogenous oestradiol on luteinizing hormone secretion in prepubertal lambs.

The effect of repeated administration of oestradiol-17beta on luteinizing hormone (LH) secretion was studied in 3-month old female lambs (n = 20). Animals were randomly divided in five groups and treated or not treated (group I) with 1, 2 or 3 i.m. injections of oestradiol in corn oil vehicle (1 microg/kg). Animals were slaughtered 12 (group II) and 24 h (group III) after the first injection, 24 h after the second injection (group IV) and 24 h after the third injection (group V). Animals in groups IV and V were catheterized and blood samples were collected every 4 h starting before treatment until time of sacrifice. In the rest of the groups blood samples were taken at the time of slaughter. The number of follicles > 1 mm in diameter on the ovarian surface were recorded and classified according to size. Maximum levels of oestradiol ranged from 103 to 250 pmol/l and returned to baseline within 12 to 16 h after each injection. LH secretion showed a consistent pattern in all lambs, with increases between 8 and 16 h after each oestradiol injection. The highest amplitude and longest duration (8-12 h) of LH secretion was recorded after the second oestradiol injection. Preliminary data indicated that FSH secretion resembled that of LH. There was an increase in the number of follicles with a diameter of more than 2 mm. Plasma concentrations of progesterone and cortisol were low and did not differ within groups or between treatments. The findings confirm that the pituitary LH release system in ewe lambs is sensitive to the stimulatory effects of oestradiol long before puberty. Results indicate that priming with oestradiol increases pituitary LH release to subsequent challenges of oestradiol, but long time exposure to oestradiol may have a negative effect on LH secretion. Although none of the oestradiol-treated lambs ovulated, the increase in the number of large follicles with repeated injections of oestradiol suggests that small follicles were gonadotrophin-responsive and stimulated by the gonadotrophin release.

Effects of estradiol and progesterone on the reproductive tract and on uterine sex steroid receptors in female lambs.

The effects of estradiol-17 beta (E(2)) and progesterone (P) on the reproductive tract and on uterine estrogen receptors and P receptors were studied in 2-mo-old female lambs (n = 11). On Days 0, 1 and 2, E(2) (1 ug/kg, Group E, n = 4), P (0.3 mg/kg, Group P, n = 4) or corn oil (control) vehicle (Group C, n = 3) were administered, and in Day 3 all lambs were slaughtered. Group E (n = 12) had E(2) serum concentrations (mean +/- SEM) of 43.8 +/- 2.2 pmol L , similar to that of the follicular phase; while P concentrations in Group P (n = 12) were similar (2.8 +/- 0.18 nmol L ) to those of the luteal phase of the ewe estrous cycle. The E(2) treatment increased the reproductive tract weight, while P treatment increased only the uterine weight. Both E(2) and P receptors from upper and middle uterine zones (including the myometrium, endometrium and caruncles) were determined by binding assays with tritiated hormones, dextran-charcoal separation and inverse Scatchard analysis. Both the E(2) and P treatments decreased E(2) and P receptor concentrations in upper and middle zones, although the upper zone had higher receptor concentrations than the middle zone (P < 0.01). E(2) receptor concentrations in the upper zone (mean +/- SEM, fmol mg prot) were 1236 +/- 34, 667 +/- 80 and 444 +/- 103 for Groups C, P and E, respectively. The P receptor concentrations were 2434 +/- 135, 1273 +/- 102 and 1536 +/- 213 for the same groups. The high uterine P receptor concentrations allowed P action without prior estrogen priming of female lambs. The present results suggest that E(2) and P might down-regulate their own and each other's receptors during development. The biological responses induced by E(2) and P, as measured by the reproductive tract weight, demonstrated that at an early stage of development uterine receptors are physiologically active.

Uterine estrogen and progesterone receptors in prepubertal ewes: distribution in myometrium, endometrium and caruncles.

In order to study the expression of uterine steroid receptors during animal development, estrogen and progesterone binding sites (E2R and P4R) were investigated in 9-month-old Corriedale ewe-lambs during the non-breeding season. E2R and P4R were identified by binding assays and Scatchard analysis in all uterine layers. The dissociation constant (Kd) values were: 0.63 +/- 0.14 nM (n = 24) for E2R and 2.10 +/- 0.32 nM (n = 24) for P4R. The E2R distribution was: 188 +/- 32, 367 +/- 83 (NS) and 494 +/- 72 fmol/mg protein (P < 0.02) for myometrium, caruncles and intercaruncular endometrium, respectively. Interestingly, high levels of P4R (fmol/mg protein) were found. By layer the P4R content was: caruncles, 1 123 +/- 198; intercaruncular endometrium, 1 283 +/- 187 (NS); and myometrium, 502 +/- 76 (P < 0.002). A positive correlation existed between both receptors (r = 0.772, P < 0.0001, n = 24). E2 and P4 circulating levels (measured by radioimmunoassay), were similar to the adult ewe basal levels. These findings suggest that ewes have sensitive and functional uterine estrogen and progesterone receptors even before cyclic ovarian activity.