Regulation and localization of insulin-like growth factor binding protein-5 gene expression in the uterus and placenta of the cyclic and early pregnant ewe

The insulin-like growth factor (IGF) system plays an important role in the regulation of uterine function and placental growth. However, there is little information regarding the localization and regulation of IGF binding protein-5 (IGFBP-5) in the reproductive tract. The distribution of this IGFBP was therefore investigated using in situ hybridization in sections of utero-placental tissue obtained throughout the estrous cycle, up to Day 55 of gestation, and on Days 16-17 from both horns of ewes with unilateral pregnancies that followed uterine transection. In nonpregnant ewes, IGFBP-5 mRNA was present at high concentrations in the maternal caruncles and luminal epithelium, and at moderate levels in myometrium. In these regions IGFBP-5 mRNA showed cyclic variations, with concentrations peaking around ovulation, whereas low expression in the endometrial stroma remained constant. During pregnancy, there was additional localization to the endometrial glands; and in all regions, with the exception of the caruncles, concentrations increased significantly with gestational age. In transected uteri, concentrations in the luminal epithelium of the pregnant horn were significantly higher than those in the nonpregnant horn. In the caruncles, IGFBP-5 mRNA formed an intense band just below the tips of the invading fetal villi. Below this band, IGFBP-5 mRNA localized to form a series of rings, which could create a route to allow the fetal villi access into the caruncular stroma for nutrient exchange. In conclusion, IGFBP-5 is abundantly expressed in the ovine reproductive tract, with both the concentration and localization differentially regulated during the cycle and pregnancy.     

Effect of a high maternal dietary intake during mid-gestation on components of the utero-placental insulin-like growth factor (IGF) system in adolescent sheep with retarded placental development

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0. 01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'-oligoadenylate synthetase expression in the ovine uterus

The enzymes which comprise the 2',5'-oligoadenylate synthetase (OAS) family are interferon (IFN) stimulated genes which regulate ribonuclease L antiviral responses and may play additional roles in control of cellular growth and differentiation. This study characterized OAS expression in the endometrium of cyclic and pregnant ewes as well as determined effects of IFNtau and progesterone on OAS expression in cyclic or ovariectomized ewes and in endometrial epithelial and stromal cell lines. In cyclic ewes, low levels of OAS protein were detected in the endometrial stroma (S) and glandular epithelium (GE). In early pregnant ewes, OAS expression increased in the S and GE on Day 15. OAS expression in the lumenal epithelium (LE) was not detected in uteri from either cyclic or pregnant ewes. Intrauterine administration of IFNtau stimulated OAS expression in the S and GE, and this effect of IFNtau was dependent on progesterone. Ovine endometrial LE, GE, and S cell lines responded to IFNtau with induction of OAS proteins. In all three cell lines, the 40/46-kDa OAS forms were induced by IFNtau, whereas the 100-kDa OAS form appeared to be constitutively expressed and not affected by IFNtau. The 69/71-kDa OAS forms were induced by IFNtau in the S and GE cell lines, but not in the LE. Collectively, these results indicate that OAS expression in the endometrial S and GE of the early pregnant ovine uterus is directly regulated by IFNtau from conceptus and requires the presence of progesterone.     

A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

Evidence suggests the insulin-like growth factor (IGF) system may be involved in luteal maintenance and regression. However, previous studies have only investigated a few components of the system, primarily in bovine and non-ruminant species. The present study investigated gene expression for the components of the IGF system in ovine corpora lutea (CL) at various key stages of the oestrous cycle (Experiment 1), and the possible regulatory effects of LH on IGF gene expression in ovine CL using a GnRH antagonist model system (Experiment 2). Experiment 1 revealed that IGF-I (P<0.001), type I (P=0.008) and II (P=0.005) IGF-Rs and IGFBP-5 (P<0.05) mRNA levels were significantly elevated in early regressing CL. In contrast, IGF-II levels were high in CL but did not vary throughout the oestrous cycle, while IGFBP-2, -3, -4 and -6 mRNA levels were highest throughout the luteal phase but lower in regressing CL (P<0.05). IGFBP-1 mRNA could not be detected in any CL. Abrogation of LH action following GnRH antagonist administration (Experiment 2) resulted in a significant increase in expression for IGF-I (P<0.001), type II IGF-R (P=0.004) and IGFBP-5 (P<0.05) after only 12h, but these increases were transient. IGF-II, type I IGF-R and IGFBP-2, -3, -4 and -6 mRNA levels remained unaffected by GnRH antagonist treatment. These data highlight the role that LH plays in regulating IGF-I gene expression and lends further support that IGF-I may be a key luteotrophic factor in sheep.     

Response of anestrous ewes to the ram effect after follicular wave synchronization with a single dose of estradiol-17beta

Anestrous ewes respond to the introduction of rams with either an ovulation within 2-3 days that may be followed by luteal phases of normal or short length, with delayed ovulations (5-6 days later), or with the luteinization of follicles. The aim of this work was to study the relationship between the growth status of the largest follicle present when rams are introduced and the type of ovarian response in non-treated ewes and in ewes treated with estradiol-17beta before ram introduction. Thirteen anestrous Corriedale ewes were divided into 2 groups: E2 (n = 7) and C (n = 6). The E2 ewes received a single dose of 50 microg estradiol-17beta 5 days before the introduction of the rams to synchronize the onset of their follicle waves, while C ewes remained untreated. When the rams were introduced, all E2 ewes had the largest follicle in a growing stage in contrast with the C ewes (3 out of 6; P < 0.05). Five C and 4 E2 ewes ovulated after the introduction of the rams (Day 3.4 +/- 0.4 for C vs. 4.8 +/- 0.3 for E2 ewes, respectively, P < 0.05). Only one ewe from each group developed a normal luteal phase: 4 C and 3 E2 ewes had short luteal phases. One C ewe and 2 E2 ewes had short luteal phases originating from follicles that did not ovulate. After the first luteal phase, all ewes returned to anesirus without a second ovulation or luteal phase. The remaining E2 ewe did not ovulate or show any changes in progesterone serum concentrations. We conclude that the growth status of the largest follicle alone does not determine the ovarian responding pattern of anestrous ewes to the ram effect.     

Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a 17-kDa protein that shows cross-reactivity with ubiquitin antisera and retains the carboxyl-terminal Leu-Arg-Gly-Gly amino acid sequence of ubiquitin that ligates to, and directs degradation of, cytosolic proteins. It has been reported that bovine endometrial UCRP is synthesized and secreted in response to conceptus-derived interferon-tau (IFNtau). In the present studies, UCRP mRNA and protein were detected in ovine endometrium. Ovine UCRP mRNA was detectable on Day 13, peaked at Day 15, and remained high through Day 19 of pregnancy. The UCRP mRNA was localized to the luminal epithelium (LE), stromal cells (ST) immediately beneath the LE, and shallow glandular epithelium (GE) on Day 13, but it extended to the deep GE, deep ST, and myometrium of uterine tissues by Day 15 of pregnancy. Western blotting revealed induction of UCRP in the endometrial extracts from pregnant, but not cyclic, ewes. Ovine UCRP was also detected in uterine flushings from Days 15 and 17 of pregnancy and immunoprecipitated from Day 17 pregnant endometrial explant-conditioned medium. Treatment of immortalized ovine LE cells with recombinant ovine (ro) IFNtau induced cytosolic expression of UCRP, and intrauterine injection of roIFNtau into ovariectomized cyclic ewes induced endometrial expression of UCRP mRNA. These results are the first to describe temporal and spatial alterations in the cellular localization of UCRP in the ruminant uterus. Collectively, UCRP is synthesized and secreted by the ovine endometrium in response to IFNtau during early pregnancy. Because UCRP is present in the uterus and uterine flushings, it may regulate endometrial proteins associated with establishment and maintenance of early pregnancy in ruminants.     

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.     

Co-expression of messenger ribonucleic acids encoding IGF-I, IGF-II, type I and II IGF receptors and IGF-binding proteins (IGFBP-1 to -6) during follicular development in the ovary of seasonally anoestrous ewes

In recent years, it has become apparent that components of the insulin-like growth factor (IGF) system are involved in the regulation of ovarian follicular development in sheep. The majority of previous studies have concentrated on investigating only a select few components and not the whole system. The aim of the present study was to use five seasonally anoestrous ewes to investigate the expression of mRNA encoding all 10 components of the sheep IGF system among various-sized follicles within the ovary, using sheep-specific ribonucleotide probes and in situ hybridisation. IGF-I mRNA expression was low and did not vary with follicle size. IGF-II mRNA expression was significantly higher (P < 0.05) in small follicles compared to large follicles. Both IGF receptors had significantly higher (P < 0.05) levels of mRNA expression in small follicles, with the type I receptor being expressed to a slightly greater extent than the type II receptor. IGFBP-2, -3, -4 and -5 gene expression followed a similar pattern to IGF-II and the IGF receptors, whereby expression decreased with increasing follicle size. Similar to IGF-I, IGFBP-6 mRNA expression showed little variation with follicle size. IGFBP-1 mRNA expression was observed at low and constant levels, albeit in small and medium-sized follicles only. These data demonstrate that all of the components of the IGF system are produced in the ovine follicle, and for some of the components, their gene expression varied with stage of follicle development. This study further emphasises the importance of IGF-II as the major IGF in the autocrine and paracrine regulation of follicle development in sheep.     

Uterine specific antigens in the ewe

Uterine flushings from ewes on days 0, 3, 6, 9, 12 and 15 of the estrous cycle were analyzed for total protein content. Flushings from days 9, 12 and 15 had greater (P<.01) amounts of protein than those from 0, 3 and 6. Antisera to uterine fluids from ewes at day 10 to 12 or day 14 to 15 of pregnancy detected two uterine-specific antigens in uterine flushings at day 7, 11 and 15 but not at days 0 and 3 of the cycle. A third uterine antigen was also detected in kidney tissue extracts. All three antigens were present in endometrial extracts at each stage examined. Progesterone, or estrogen plus progesterone, administration to ovariectomized ewes induced the appearance of the two uterine-specific antigens. The third antigen was detectable even in ovariectomized ewes. No pregnancy-specific antigens were detected in flushings from days 7, 11 or 15 of gestation. The effect of pregnancy on endometrial protein synthesis was examined in vitro . No differences were seen in the incorporation of (3)H-leucine in day 11 pregnant or nonpregnant or in day 14 pregnant or nonpregnant endometrium. No differences in total uterine lumenal protein were observed. Endometrial secretions, obtained by conditioning media with endometrial explant cultures, were evaluated to assess their effect on protein synthesis in day 11 embryos cultured in vitro . No significant effects of endometrial secretions or serum were observed.     

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

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

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.     

Follicular and luteal expression of insulin-like growth factors I and II and the type 1 IGF receptor in the bovine ovary.

The localization of mRNAs for insulin-like growth factors I (IGF-I) and II (IGF-II) and the type 1 IGF receptor (IGF-1R) in bovine follicles and corpora lutea was determined using in situ hybridization on sectioned ovaries collected from nonpregnant, cyclic Holstein cows in either the follicular (n = 3) or luteal (n = 5) phases of the cycle. Concentrations were measured as absorbance units of individual regions or follicles from autoradiographs. There was intense follicular expression of mRNAs encoding IGF-II and IGF-1R. For mRNA encoding IGF-II, expression was significantly higher in smaller follicles (< 5 mm diameter, P < 0.01) and, in this size range, expression was significantly greater in healthy compared with atretic follicles (P < 0.01). For mRNA encoding IGF-1R, there was no effect of size but concentrations were again significantly greater in healthy compared with atretic follicles of < 5 mm. In medium (5-10 mm) and large (> 10 mm) follicles, there was no effect of health for expression of either IGF-II or IGF-1R. mRNA encoding IGF-II was found exclusively in the theca, whereas mRNA encoding IGF-1R was confined to the granulosa layer. IGF-I expression was not detectable in 83% of the 53 follicles examined. In the remaining 17% of follicles, expression was very low and was unrelated to size or state of atresia. mRNAs encoding IGF-I, -II and IGF-1R were all present in the corpus luteum, whereas only those for IGF-II and IGF-1R were found in ovarian stroma. These data indicate that the insulin-like growth factors play a significant role in follicular and luteal development in the bovine ovary. Locally produced IGF-II is probably an important regulator of follicular growth, whereas most of the IGF-I present in follicular fluid is likely to be derived from the circulation.     

Ultrasound and endocrine evaluation of the ovarian response to a single dose of 500 IU of eCG following a 12-day treatment with progestogen-releasing intravaginal sponges in the breeding and nonbreeding seasons in ewes

A standard dose of 500 IU of eCG is commonly given to progestogen pre-treated anestrous ewes for induction of estrus. Twelve seasonally anestrous and 12 cyclic Western White Face ewes were treated for 12 days with intravaginal sponges impregnated with medroxyprogesterone acetate (MAP). In trials in both the breeding and nonbreeding seasons, six randomly selected ewes were given 500 IU of eCG at sponge removal to determine the effects of low dose of eCG on ovarian antral follicular dynamics and ovulation. Ultrasound scanning and blood sampling were done daily. Treatment with eCG did not have marked effects on antral follicular growth. All ewes ovulated, except for five of six control anestrous ewes. Luteal structures and progesterone secretion were confirmed in all but the control anestrous ewes. In the breeding season, peak progesterone concentrations were greater (P<0.05) in eCG-treated compared to control ewes. Daily serum estradiol concentrations were greater in the periovulatory period in eCG-treated compared to control ewes (treatment-by-day interaction; P<0.05), particularly in anestrus. Progestogen-treated ewes ovulated follicles from several follicular waves, in contrast to ovulations of follicles from the final wave of the cycle in untreated, cyclic ewes. Anestrous ewes exhibited more frequent follicular waves and FSH peaks compared to cyclic ewes after a progestogen/eCG treatment. In conclusion, 500 IU of eCG given after 12 days of progestogen treatment had limited effects on the dynamics of ovarian follicular waves. However, eCG treatment increased serum concentrations of estradiol during the periovulatory period, particularly in anestrous ewes; this probably resulted in the synchronous estrus and ovulation in anestrous ewes.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.