Uterine Milk Proteins Research Articles

Role of preovulatory concentrations of estradiol on timing of conception and regulation of the uterine environment in beef cattle

ABSTRACTThe ability to induce ovulation with an injection of GnRH facilitated the development of fixed-time artificial insemination (AI) protocols. However, animals that exhibit estrus prior to fixed-time AI have greater pregnancy success. Thus, the objectives of the present experiments were to determine the impact of estrus expression prior to fixed-time AI on timing of conception and to characterize the role of preovulatory estradiol in regulating changes in expression of uterine genes. In experiment 1, data were collected from 4,499 beef cows inseminated by fixed-time AI in 31 different herds. Animals that did not conceive to AI but exhibited estrus before timed-AI were more likely to conceive during cycle 1 after AI, and overall conceived earlier in the breeding season compared to animals that did not exhibit estrus. In experiment 2, beef cows were synchronized using a fixed-time AI protocol. Uterine horn biopsies and blood samples were collected on Day 0, 5, 10, or 16. Concentrations of estradiol on Day 0 did not influence expression of progesterone receptor, ER beta, or oxytocin receptor. Increased concentrations of estradiol on Day 0 increased expression of ER alpha from Days 0 to 5 of the estrous cycle. Furthermore, cows with increased concentrations of estradiol on Day 0 had increased expression of inhibin beta A, and uterine milk protein precursor. Thus, animals that do not exhibit estrus prior to fixed-time AI had decreased breeding season pregnancy success and conceived later in the breeding season, and preovulatory concentrations of estradiol likely play a major role in this establishment of pregnancy, not only directly by regulating uterine gene expression, but also indirectly throughout the subsequent estrous cycle.

Placental endocrine malfunction programs ovarian defects in female murine offspring

Development of the placenta must always be in advance of that of the embryo. Evidence from domestic species demonstrates that the placenta is capable of stimulating its own development through a signalling dialogue with the endometrial glands. Placental lactogens produced by the trophoblast lead to increased expression and release of uterine secretions and mitogenic growth factors, including epidermal growth factor, that have a close temporal and spatial relationship with trophoblast proliferation. Here, we review evidence that an equivalent mechanism operates in the human. The same repertoire of receptors is present on the endometrial gland cells, and the epithelial cells have long been known to adopt a hypersecretory phenotype following an implantation. Furthermore, early pregnancy hormones stimulate the secretion of glycodelin-A and osteopontin, two ‘uterine milk proteins’ that have multiple potential effects at the maternal-placental interface, from organoid cultures derived from endometrial glands. Prolactin appears to be an important stimulant, but unlike in domestic species the human trophoblast does not secrete this hormone. Instead, it is a major product of decidual cells. Hence, complications of pregnancy that have their pathophysiological roots in deficient trophoblast proliferation may be due primarily to problems of decidualisation. Ensuring the endometrium is in an optimal state pre-conceptionally should therefore be a priority for women's health. Trophoblast stemness and proliferative capacity show a sharp decline at the switch from histotrophic to haemotrophic nutrition. This may reflect the increase in oxygen concentration or loss of growth factor support. Either way, there are implications for adaptive growth of the organ.

Interactions between the immune system and the ruminant conceptus

Interactions of the conceptus with the immune system can involve either anti-sperm or anti-conceptus immune responses that limit the success of pregnancy of beneficial effects of cytokines released from lymphoid cells on embryonic growth and gene expression. The immune system is functional in the uterus and therefore there is the potential for anti-conceptus immune responses. However, endometrial lymphocytes are distinct in many respects from lymphoid cells at peripheral sites; one major subpopulation expresses the gamma delta T-cell receptor and may not recognize major histocompatibility antigens. There are also several control systems to limit anti-conceptus immune responses. In particular, expression of major histocompatibility antigens on the trophoblast is either absent or of limited distribution. In addition, activation of anti-conceptus immune responses leading to cytolytic responses is further limited by the presence of molecules that can inhibit lymphocyte transformation. The most well-characterized of these are prostaglandin E2 from placental and endometrial tissues, interferon-tau from the trophoblast during early pregnancy, and two endometrial proteins called the uterine milk proteins (UTMP). Progesterone plays a central role in inhibition of immune responses in actions that are mediated at least in part through endometrial secretion of UTMP. Cytokines play important roles as autocrine and paracrine regulators in many tissues including the reproductive tract. In ruminants, the best described example is interferon-tau. Other cytokines found in the reproductive tract or produced by the conceptus include interleukin-1, leukaemia inhibitory factor, granulocyte-macrophage colony stimulating factor and interleukin-6. It is possible that the major source of cytokines in the reproductive tract is non-lymphoid cells of the endometrium and trophoblast. It is not known to what extent endometrial lymphocytes contribute to the cytokine milieu because no cytokine has been identified as a product of endometrial lymphocytes. However, there is a population of granulated lymphocytes that increase in number and granularity in the luminal epithelium of the late-pregnant ewe that is a potential source of cytokines.

Reactive Oxygen Species May Be Involved in the Signaling of Equine Sperm Chemotaxis

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

The Effect of Buserelin on Reproductive Performance in the Transitional and Anestrous Mare

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

Evaluation of the Effect of Seminal Plasma in the Post Insemination Uterine Inflammatory Dynamic by Power Doppler Ultrasonography and Low Volume Lavage in the Mare

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

Evaluation of Cooled-storage After Thawing Frozen Stallion Semen

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

Concentrations of Gentamicin In Serum, Intrauterine Fluid, and Endometrial Tissue After Intravenous Administration in Healthy Mares

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

Filtration of Commercial Extenders Prior to Refreezing Stallion Sperm for ICSI

In all eutherian mammals, there are two modes for supplying nutrients to the conceptus, histotrophic and hemotrophic nutrition. In general, these modes are sequential; although in some species, the two operate in parallel throughout the entire pregnancy. Although hemotrophic nutrition is the most important for proper growth of the fetus as assessed by birth weight, there is growing evidence that the phase of histotrophic nutrition is critical for initial development of the placenta. Evidence from animal species has revealed the existence of a signaling dialog between the trophoblast of the placenta and the endometrial glands, whereby the secretion of nutrients, collectively known as uterine milk proteins, and growth factors is upregulated during early pregnancy. In this way, the placenta is able to stimulate its own development. Circumstantial evidence suggests that an equivalent dialog occurs in the human and that deficiencies in endometrial function in early pregnancy may underpin complications of later pregnancy. The conservation of the trophoblast-endometrial dialog across species, with such differences in placental types as the horse and the human, suggests that it is of fundamental importance in placentation. The implication is that attempts should be made to ensure the endometrium is in optimal condition before conception.

The Uterine Milk Protein (UTMP) gene is associated with milk production traits in Iranian Holstein bulls

Uterine Milk Protein (UTMP) the member of Serine Protease Inhibitor superfamily secreted from uterine endometrium during pregnancy under influence of progesterone. Specific functions of UTMP include protease inhibition, growth control, and direct nutrition of the conceptus. This investigation was performed in order to study polymorphism of UTMP gene and its association with milk production traits in Iranian Holstein bulls. Genomic DNA was extracted from 100 semen samples of proven bulls. In order to amplify 568bp-fragment including same part of intron 3, whole exon 4 and downstream of UTMP gene, a pair of primer was designed. PCR products were digested with BsrI enzyme. The allele frequencies of a, b and c were 0.135, 0.69 and 0.175, respectively in studied population. Four genotypes AB, BB, BC and CC were observed with frequencies of 0.27, 0.49, 0.13 and 0.11, respectively. Genotypes AA and AC were not observed in this study. The chi-square (X2) test revealed deviation from Hardy-Weinberg equilibrium. Data were obtained from animal breeding centre of Iran for the first lactation during 1993-2008 to estimate some genetic parameters. Heritability of milk yield, fat yield, protein yield, fat percent and protein percent were 0.28, 0.21, 0.22, 0.32 and 0.34 respectively. Results indicated significant association between UTMP Genotypes and milk, fat and protein yield traits. Meanwhile, Bulls with CC Genotype had higher milk, fat and protein yield compared to other genotypes (p<0.05). These results suggested UTMP as a candidate gene influencing milk production traits might be implemented in breeding programs to improve the production performance of Iranian Holstein cattle.

Changes in expression of cell‐cycle‐related genes in PC‐3 prostate cancer cells caused by ovine uterine serpin

The hormonal-regulated serpin, ovine uterine serpin (OvUS), also called uterine milk protein (UTMP), inhibits proliferation of lymphocytes and prostate cancer (PC-3) cells by blocking cell-cycle progression. The present aim was to identify cell-cycle-related genes regulated by OvUS in PC-3 cells using the quantitative human cell-cycle RT(2) Profiler PCR array. Cells were cultured +/-200 microg/ml recombinant OvUS (rOvUS) for 12 and 24 h. At 12 h, rOvUS increased expression of three genes related to cell-cycle checkpoints and arrest (CDKN1A, CDKN2B, and CCNG2). Also, 14 genes were down-regulated including genes involved in progression through S (MCM3, MCM5, PCNA), M (CDC2, CKS2, CCNH, BIRC5, MAD2L1, MAD2L2), G(1) (CDK4, CUL1, CDKN3) and DNA damage checkpoint and repair genes RAD1 and RBPP8. At 24 h, rOvUS decreased expression of 16 genes related to regulation and progression through M (BIRC5, CCNB1, CKS2, CDK5RAP1, CDC20, E2F4, MAD2L2) and G(1) (CDK4, CDKN3, TFDP2), DNA damage checkpoints and repair (RAD17, BRCA1, BCCIP, KPNA2, RAD1). Also, rOvUS down-regulated the cell proliferation marker gene MKI67, which is absent in cells at G(0). Results showed that OvUS blocks cell-cycle progression through upregulation of cell-cycle checkpoint and arrest genes and down-regulation of genes involved in cell-cycle progression.

Association Between Preovulatory Concentrations of Estradiol and Expression of Uterine Milk Protein Precursor, Inhibin Beta A, and Proenkephalin.

Eliminating the preovulatory surge of estradiol decreased embryo survival following embryo transfer, uterine weight, uterine protein, RNA to DNA ratio, and the rate of protein synthesis in sheep. Furthermore, cows that did not exhibit standing estrus (decreased preovulatory concentrations of estradiol) around the time of gonadotropin-releasing hormone (GnRH)-induced ovulation had decreased pregnancy success compared to cows that did have elevated preovulatory concentrations of estradiol. Thus, induction of ovulation alone may not be sufficient for maximum pregnancy success. Uterine environment, as influenced by endometrial protein expression, plays a central role in early embryo development and survival. Therefore, the objective of the present experiment was to characterize changes in expression of uterine milk protein precursor, inhibin beta A, and proenkephalin between cows with high (highE2; n = 6) and low (lowE2; n = 5) concentrations of estradiol at time of GnRH induced ovulation (day 0). Beef cows were treated with 100 μg GnRH on day -9, 25 mg PG on day -2, and 100 μg GnRH on day 0. Uterine horn biopsies (collected ipsilateral to the ovulatory follicle) and blood samples were collected on days 0 and 16. Total cellular RNA was extracted from all biopsies and relative mRNA levels for uterine milk protein precursor, inhibin beta A, proenkephalin, and polyubiqutin were determined by real-time RT-PCR and corrected for polyubiqutin. Circulating concentrations of estradiol on day 0 were greater (P < 0.001) in highE2 cows (8.33 ± 1.13 pg/mL) compared to lowE2 cows (2.72 ± 1.24 pg/mL). Relative expression of uterine milk protein precursor was greater (P = 0.04) in highE2 cows compared to lowE2 cows on day 0 (12.51 ± 3.07 vs. 2.10 ± 3.37, respectively). However, expression decreased (P = 0.002) from day 0 to day 16 in highE2 cows, and there was no difference (P = 0.95) in uterine milk protein precursor expression between highE2 and lowE2 cows on day 16 (1.11 ± 0.21 vs. 1.13 ± 0.34, respectively). Relative expression of inhibin beta A was greater (P = 0.03) in highE2 cows compared to lowE2 cows on day 0 (8.81 ± 1.64 vs. 2.82 ± 1.80, respectively), and expression decreased (P = 0.002) in highE2 cows from day 0 to 16. On day 16, there was no difference (P = 0.74) in inhibin beta A expression between highE2 and lowE2 cows (0.28 ± 0.03 vs. 0.29 ± 0.05, respectively). Expression of proenkephalin was not detectable in either highE2 or lowE2 cows on day 0. However, relative expression of proenkephalin on day 16 was greater (P = 0.02) in highE2 cows (10.40 ± 1.95) compared to lowE2 cows (2.70 ± 2.49). In summary, preovulatory concentrations of estradiol regulated uterine expression of uterine milk protein precursor, inhibin beta A, and proenkephalin during the subsequent estrous cycle. Reduced fertility among cows with decreased concentrations of estradiol prior to GnRH-induced ovulation may be associated with effects of changes in uterine protein expression on uterine environment.

Pattern of Expression of the Uterine Milk Protein Gene and its Association with Productive Life in Dairy Cattle

The uterine milk proteins (UTMP) are the major proteins secreted by the endometrium, primarily under the control of progesterone. Specific functions of UTMP are poorly understood, but may include protease inhibition, nutrition of the conceptus, growth control, and suppression of the maternal immune system. The uterine milk protein gene (UTMP) was chosen for this study because of its possible roles in health traits and results of previous studies on the association of the UTMP region with milk production and productive life in dairy cattle. Expression of UTMP was examined in 198 bovine tissues obtained from 10 fetuses and 17 cows. Transcripts of UTMP were found in all cotyledon tissues examined and to a lesser extent in ovary, pituitary, and spleen tissues obtained from fetuses. The UTMP gene was predominantly expressed in endometrium (17/17), ovary (15/16), and caruncle (12/12) tissues obtained from cows. The predominant expression of UTMP in reproductive tissues is consistent with an important role of this gene in reproductive success. To investigate the association between UTMP and production traits in cattle, we identified 2 synonymous single nucleotide polymorphisms (SNP) at positions 1179 (A/G) and 1296 (A/G) using the pooled DNA sequencing approach. The DNA was extracted from 28 Holstein sires and their 1,362 sons obtained from the Cooperative Dairy DNA Repository and from 913 cows from the University of Wisconsin resource population. Single nucleotide polymorphism 1296 was associated with a significant increase in productive life in both populations. This finding is similar to results previously obtained for the protease inhibitor gene, which is near UTMP and was also found to be associated with productive life in dairy cattle. Differential allelic expression of UTMP was observed in reproductive tissues obtained from 9 heterozygous individuals. The differential allelic expression observed in this study is consistent with other studies showing a correlation between allelic variation in gene expression and phenotypic variability.

172 DOES ESTRADIOL-17β CAUSE CYCLE-DEPENDENT MODULATIONS OF UTERINE MILK PROTEIN IN BOVINE ENDOMETRIUM?

Uterine secretion from the endometrial glandular epithelium provides optimal conditions for early embryonic development. The uterine milk protein (UTMP), a member of the serine proteinase inhibitor superfamily, has been demonstrated to be a major progesterone-induced glycoprotein secreted by the endometrium during pregnancy. Previous transcriptomic analysis revealed that UTMP was highly abundant at estrus in the bovine endometrium (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889–908). Here we describe a detailed characterization of UTMP mRNA expression at several time points during the bovine estrous cycle and the pre-implantation period. Simmental heifers were monitored with respect to serum progesterone (P4) and estradiol-17β (E2), and slaughtered at estrus or 3.5, 12, 15, or 18 days after estrus, or at Day 15 or 18 of pregnancy (n = 4 per group). The uterus was divided into corpus and caudal, middle and cranial parts of the ipsilateral uterine horn for sampling of intercaruncular endometrium. In addition, effects of steroid hormones were investigated by stimulating an endometrial cell culture obtained from Day 8 animals (n = 4) with physiological doses of P4 or E2. In all cases, UTMP mRNA was quantified by real-time RT-PCR. Pronounced changes of UTMP mRNA abundance were detected during the estrous cycle. Expression was highest at estrus, followed by a remarkable decrease at Day 3.5. There was no difference between pregnant and non-pregnant animals at Day 15. Cycling animals displaying a high P4 and low E2 content (P4 &amp;gt; 2 ng mL-1 and E2 &amp;lt; 1 pg mL-1) revealed a lower expression of UTMP at Day 18 compared to the pregnant heifers, whereas animals at Day 18 progressing toward estrus (P4 &amp;lt; 1.5 ng mL-1 and E2 &amp;gt; 3 pg mL-1) exceeded the mRNA expression of the pregnant group. Following stimulation with estradiol-17β, the in vitro UTMP transcripts increased significantly. These results indicate that two different interfering stimulatory events might take place. While estradiol-17β appeared to increase UTMP mRNA expression at estrus, a second factor, most probably embryo-derived or embryo-induced, is assumed to be responsible for the UTMP rise during early pregnancy. The distinct gradient from the cranial uterine horn to the corpus at estrus was less pronounced at Day 3.5 and absent at Days 12, 15, and 18, pointing toward functional implications regarding the passing gametes, particularly sperm. An antibody raised against bovine UTMP will further validate the observed mRNA regulations on the protein level. In conclusion, bovine UTMP seems to play a decisive role for precise cyclic regulation of the bovine uterine milieu and during early embryo-maternal communication. This work was supported by the DFG FOR 478.

Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes

The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle--end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-beta signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

211 TEMPORAL AND SPATIAL EXPRESSION PROFILE OF THE UTERINE MILK PROTEIN — A MEMBER OF THE SERINE PROTEASE INHIBITOR SUPERFAMILY — IN THE BOVINE ENDOMETRIUM

A combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify differentially expressed genes in the bovine intercaruncular endometrium at estrus and diestrus. Six cyclic Simmental heifers were cycle-synchronized and slaughtered the morning after standing heat occurred (n = 3) or 12 days after estrus (n = 3), respectively. The uterus was divided into seven sections: corpus, plus the caudal, middle, and cranial parts of both uterine horns. Samples were collected from the epithelial layer of the lamina propria of the intercaruncular endometrium. Two subtracted libraries were prepared and analyzed by array hybridization with probes produced from the 42 tissue samples. More than 130 differentially expressed genes were identified. Among these genes, the mRNA for the uterine milk protein (UTMP) showed a much higher expression level at estrus versus diestrus. Furthermore, the expression level differed markedly between the uterus sections. Statistical significance was tested using ANOVA and Student Newman Keuls post-hoc test (GeneSpring® version 6.1, Silicon Genetics, Redwood City, CA, USA). There was a pronounced expression gradient from both cranial uterine horns (highest) to the corpus (lowest) at estrus. UTMP mRNA level was highest in the ipsilateral cranial horn. A more accurate quantification was done by quantitative real-time RT-PCR using the same RNA samples as for array hybridization and, in addition, samples from further time points of the estrous cycle and early pregnancy. The results obtained by array hybridization were clearly confirmed. UTMPs, also known as uterine serpins (US), were previously shown to be the major products secreted by the uteri of cattle, sheep, and pigs during pregnancy. The US are glycoproteins belonging to the serpin (serine proteinase inhibitor) superfamily of proteins. However, no inhibitory activity to any serine proteinase has been found. The ovine uterine serpin (OvUS) has been shown to inhibit a wide variety of immune responses, including mixed lymphocyte reaction, T cell-dependent antibody production, and NK (natural killer)-like activity, indicating a major role in protecting the conceptus from maternal immune reactions. The four members of the US family form a clade distinct from other serpins, which have probably adopted important biological functions in the reproductive biology of Artiodactyla. Therefore, the identified expression gradient of the UTMP mRNA at estrus may be of particular importance, e.g. for sperm selection, and deserves further investigation at the protein level and in functional studies. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).

Effect of maternal body condition on placental and fetal growth and the insulin-like growth factor axis in Dorset ewes

This study investigated the effects of maternal body condition on fetal growth. Fetal and placental parameters from Dorset ewes of body condition score 2.0 (lean, n = 5), 3.5 (moderate, n = 7) and 5.0 (fat, n = 4) at mating were studied on day 65 of gestation. The fetal weight and fetal weight:crown-rump length ratio were greater in fat ewes than in ewes of moderate condition. The raised total and mean placentome weight in fat ewes compared with ewes of moderate condition may have contributed to their increased fetal growth. However, the fetal crown-rump length was not affected. With in situ hybridization, insulin-like growth factor II (IGF-II) mRNA and insulin-like growth factor binding protein 2 (IGFBP-2), -3 and -6 were all detected in the placentome capsule; IGF-II mRNA was also found in the mesoderm of the fetal villi and IGFBP-3 and IGFBP-6 were present in the caruncular stroma of the maternal villi. Ewes of moderate condition, which had the smallest placentae, had the greatest placental expression of IGF-II, IGFBP-2 and IGFBP-3. In the intercotyledonary endometrium, IGFBP-3, IGFBP-5 and uterine milk protein (UTMP) mRNA were all expressed in the glandular epithelium. IGFBP-3 and IGFBP-5 absorbance values were lowest in the lean ewes, whereas UTMP values were highest. Maternal insulin concentrations were greater in fat ewes, whereas plasma glucose and IGF-I concentrations in the fetal compartment were lowest in fat ewes. Therefore, in obese ewes, fetal and placental growth is increased in mid-gestation in association with higher maternal insulin concentrations and lower expression of IGFBPs in the maternal placentomes. Placental and fetal development in lean ewes may be promoted by reduced IGFBP expression in the placentomes and enhanced UTMP production by the endometrial glands. The ewes of moderate condition had the smallest fetuses and placentae coupled with the highest placental expression of IGF-II and IGFBPs.

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.

Postpartum uterine involution in sheep: histoarchitecture and changes in endometrial gene expression

After parturition, the uterus undergoes marked remodelling during involution; however, little is known of the hormonal, cellular and molecular mechanisms that regulate this process. The working hypothesis used in this study is that return of the ovine uterus to a non-pregnant state involves termination of a hormonal servomechanism that regulates endometrial gland morphogenesis and function during pregnancy. Suffolk ewes were ovariohysterectomized on postpartum days 1, 7, 14 or 28. Serum concentrations of oestradiol were high at parturition, declined to postpartum day 4, peaked on postpartum day 6, and then declined and remained low thereafter. Progesterone was undetectable in plasma from ewes post partum. Uterine wet mass and horn length decreased after postpartum day 1, but ovarian mass did not change. Residual placental cotyledons were present in the maternal caruncles on postpartum days 1 and 7 and were extruded by postpartum day 14 as plaques that were resorbed by postpartum day 28. The width of the total endometrium, stratum compactum, stratum spongiosum and myometrium, as well as endometrial gland density, decreased after parturition. Most apoptotic cells in the involuting uterus were large, vacuolated and located between the endometrial glandular epithelial cells on postpartum days 1 and 7. Immunofluorescence analyses identified both T and B cells within the glandular epithelium on postpartum day 1. Cell proliferation was detected in the luminal epithelium and glandular epithelium on postpartum days 1 and 7. On postpartum day 1, expression of oestrogen receptor alpha (ERalpha) was not detected in luminal epithelium and was low in glandular epithelium, but ERalpha was present in epithelia thereafter. Progesterone receptor (PR) protein was not detected in endometrial epithelia on postpartum day 1, but was detected in the glandular epithelium thereafter. Between postpartum days 1 and 7, ERalpha and PR protein increased substantially in the endometrial glandular epithelium. On postpartum days 1-28, abundant expression of oxytocin receptor mRNA was detected in endometrial luminal epithelium and superficial to the middle glandular epithelium. Prolactin receptor (PRLR) mRNA was detected in glandular epithelium on all postpartum days, whereas mRNA for uterine milk protein (UTMP), an index of secretory capacity of glandular epithelium, was present only on postpartum day 1. Collectively, these results indicate that uterine involution in ewes involves remodelling of both caruncular and intercaruncular areas of the uterine wall and termination of differentiated uterine gland functions characteristic of pregnancy.

Postpartum uterine involution in sheep: histoarchitecture and changes in endometrial gene expression

After parturition, the uterus undergoes marked remodelling during involution; however, little is known of the hormonal, cellular and molecular mechanisms that regulate this process. The working hypothesis used in this study is that return of the ovine uterus to a non-pregnant state involves termination of a hormonal servomechanism that regulates endometrial gland morphogenesis and function during pregnancy. Suffolk ewes were ovariohysterectomized on postpartum days 1, 7, 14 or 28. Serum concentrations of oestradiol were high at parturition, declined to postpartum day 4, peaked on postpartum day 6, and then declined and remained low thereafter. Progesterone was undetectable in plasma from ewes post partum. Uterine wet mass and horn length decreased after postpartum day 1, but ovarian mass did not change. Residual placental cotyledons were present in the maternal caruncles on postpartum days 1 and 7 and were extruded by postpartum day 14 as plaques that were resorbed by postpartum day 28. The width of the total endometrium, stratum compactum, stratum spongiosum and myometrium, as well as endometrial gland density, decreased after parturition. Most apoptotic cells in the involuting uterus were large, vacuolated and located between the endometrial glandular epithelial cells on postpartum days 1 and 7. Immunofluorescence analyses identified both T and B cells within the glandular epithelium on postpartum day 1. Cell proliferation was detected in the luminal epithelium and glandular epithelium on postpartum days 1 and 7. On postpartum day 1, expression of oestrogen receptor alpha (ERalpha) was not detected in luminal epithelium and was low in glandular epithelium, but ERalpha was present in epithelia thereafter. Progesterone receptor (PR) protein was not detected in endometrial epithelia on postpartum day 1, but was detected in the glandular epithelium thereafter. Between postpartum days 1 and 7, ERalpha and PR protein increased substantially in the endometrial glandular epithelium. On postpartum days 1-28, abundant expression of oxytocin receptor mRNA was detected in endometrial luminal epithelium and superficial to the middle glandular epithelium. Prolactin receptor (PRLR) mRNA was detected in glandular epithelium on all postpartum days, whereas mRNA for uterine milk protein (UTMP), an index of secretory capacity of glandular epithelium, was present only on postpartum day 1. Collectively, these results indicate that uterine involution in ewes involves remodelling of both caruncular and intercaruncular areas of the uterine wall and termination of differentiated uterine gland functions characteristic of pregnancy.