secretion of the major progesterone-induced proteins of the sheep uterus by caruncular and intercaruncular endometrium of the pregnant ewe from days 20–140 of gestation

An experiment was performed to evaluate the types and quantity of proteins secreted by intercaruncular endometrium at days 20, 60, 100 and 140 of gestation and caruncular endometrium from days 100 and 140 of gestation. Tissues were obtained from ewes made unilaterally pregnant. Based on SDS polyacrylamide gel electrophoresis (SDS-PAGE), the major proteins present in uterine fluid at days 60–140 of gestation were the uterine milk proteins (UTM-proteins), a pair of structurally-related, progesterone-induced polypeptides with molecular weights of 55,000 and 57,000. These proteins were also present in uterine fluid at day 20, but the major protein at this time migrated coincident with albumin. Cultured explants of endometrium at all days of pregnancy produced UTM-proteins as their major radiolabelled product for both caruncular and intercaruncular endometrium. The amount of protein secretion in vitro was greater (P<.04) for intercaruncular endometrium than for caruncular endometrium but was not significantly affected by stage of gestation or local presence of the conceptus. Immunohistochemical analysis revealed that UTM-proteins were present in glandular and luminal epithelium of intercaruncular endometrium and in both epithelial and stromal elements of caruncular endometrium. It was concluded that the UTM-proteins are produced earlier than previously described (i.e., day 20). In addition, caruncles contribute to the uterine secretory protein milieu through the secretion of proteins that are similar to that produced by the glandular intercaruncular epithelium.     

Production of calcium maintenance factor Stanniocalcin-1 (STC1) by the equine endometrium during the early pregnant period

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 β (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.     

Structure and Steroidogenesis of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.     

Mammary gland secretory concretions contain non-collagenous bone matrix proteins

Secretory concretions in mammary gland alveoli are commonly of microscopical size. However, some concretions reach clinically palpable dimensions and may occlude teat canals and obstruct milk flow. We studied secretory concretions in sheep, goat and cow mammary glands, using routine histological staining methods, conventional histochemistry and electron microscopy. As concretions frequently mineralize, immunostaining for keratan sulphate and calcium-binding non-collagenous bone matrix proteins (bone sialoprotein, osteocalcin, osteonectin and osteopontin) was performed. Concretions consisted of organic matrix (condensed secretions) with calcium precipitates. Mineralized deposits mostly show concentric organization, bound haematoxylin, and were readily identified in H&E-stained sections. Mineral components of concretions reacted for calcium carbonate and phosphate, organic matrix was found to contain sialoglycan material. Immunohistochemistry revealed bone sialoprotein, osteonectin and keratan sulphate in cow and goat concretions. Osteocalcin was detected in sheep, cow and goat concretions, whilst osteopontin was not identified in any of the specimens studied. Our results indicate the presence of non-collagenous bone matrix proteins (except osteopontin) in mammary gland concretions. These glycoproteins are commonly thought to govern mineralization of organic matrix and are assumed also to promote mineral deposition in mammary gland secretory concretions. Besides caseins, these particular glycoproteins have to be considered as calcium-binding milk proteins.     

Expression of oestrogen receptor,androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.     

妊娠早期蒙古绵羊子宫内膜enJSRV及IFN-τ基因表达水平的分析

本研究旨在检测内源性绵羊肺腺瘤反转录病毒(enJSRV)与干扰素-τ(IFN-τ)在妊娠早期蒙古绵羊子宫内膜组织的表达以及enJSRV的表达与外周血孕酮水平变化的关系。运用TaqMan实时荧光定量PCR技术和电化学发光法对enJSRV和IFN-τ在妊娠早期蒙古绵羊子宫内膜组织相对表达及孕酮水平进行了测定。实时荧光定量PCR结果显示,enJSRV和IFN-τmRNA在妊娠早期绵羊子宫内膜组织有不同程度的表达。SAS统计学软件分析得出,子宫内膜组织中enJSRV mRNA在妊娠12～14d(交配日为0d)表达较高,2～10、16～30d的表达均低于前者。IFN-τmRNA仅在妊娠12～25d表达,14d达其峰值,30d就不能检出,且差异都极显著(P<0.01)。电化学发光法结果显示,孕酮水平在妊娠2d为0.4ng·mL-1,以后升高,妊娠8～16d维持在8.3ng·mL-1左右,在妊娠19～30d孕酮水平有所下降,30d为2.5ng·mL-1。以上结果提示,子宫内膜组织中enJSRV mRNA的表达与外周血孕酮含量及IFN-τ的变化高度相关,且enJSRV在胎盘的形态发生及生殖生物学方面发挥重要作用。     

Identification of the antigenic proteins of Cowdria ruminantium.

Immunoblotting of Cowdria ruminantium proteins with sheep or bovine antiserum identified 2 antigenically conserved proteins, one being an immunodominant 31 kDa and the other a minor 27 kDa protein. These proteins are present in the electrophoretic profiles of the Welgevonden, Ball 3 and Kwanyanga stocks and are recognized by sheep antiserum to the Welgevonden, Ball 3, Kwanyanga, Mali, Comoro, Breed, Germishuys, Kümm and Mara stocks and by bovine antiserum to the Welgevonden stock of C. ruminantium. The stocks did not reveal identical or unique antigenic properties which could explain differences in pathogenicity and cross-immunity observed amongst the various stocks of C. ruminantium.     

Occurrence of ovine herpesvirus type-2 infection in sheep and cattle in Samsun Province, Turkey

July 2004, a cow with clinical signs of ovine herpesvirus type-2 infection which is known as sheep associated malignant catarrhal fever (SA-MCF) was reported in Samsun Province in Turkey. Blood samples were collected from the suspected cow, 10 sheep housed with it, and from 150 healthy sheep and 29 healthy cattle randomly selected from different places in Samsun Province. Nested polymerase chain reaction (n-PCR) was used to detect ovine herpesvirus type-2 (OvHV-2) DNA in the suspected cow and competitive- ELISA (c-ELISA) kits were used to detect antibodies against OvHV-2. The suspected cow was found to be n-PCR positive and c-ELISA negative. The serological results were as follows: All 10 (100%) of sheep housed with the suspected cow and 18 of 29 (62%) of the randomly selected cattle were found seropositive. All 150 randomly selected healthy sheep were seronegative. The overall percentage of seropositivity was 14.7% (28/190). OvHV-2 DNA was detected in the peripheral blood leucocyte (PBL) samples of the cow and of the 10 sheep housed with the suspected cow.     

The potential function of endometrial‐secreted factors for endometrium remodeling during the estrous cycle

Uterine has a pivotal role in implantation and conceptus development. To prepare a conducive uterine condition for possibly new gestation during the estrous cycle, uterine endometrium undergoes dramatic remodeling. In addition, angiogenesis is an indispensable biological process of endometrium remodeling. Furthermore, essential protein expressions related to important biological processes of endometrium remodeling, which are vascular endothelial growth factor (VEGF), myoglobin (MYG), collagen type IV (COL4), fucosyltransferase IV (FUT4), and cysteine‐rich protein 2 (CRP2), were detected in the endometrial tissue reported in many previous studies and recently discovered in histotroph substrates during the estrous cycle. Those proteins, which are liable for provoking new vessel development, cell proliferation, cell adhesion, and cell migration, were expressed higher in the histotroph during the luteal phase than follicular phase. Histotroph proteins considerably contribute to endometrium remodeling during the estrous cycle. To that end, the following review will discuss and highlight the relevant information and evidence of the uterine fluid proteins as endometrial‐secreted factors that adequately indicate the potential role of the uterine secretions to be involved in the endometrial remodeling process.     

Beta-carotene uptake and changes in ovarian steroids and uterine proteins during the estrous cycle in the canine

The uptake of beta-carotene by reproductive tissues and the effects of beta-carotene on reproductive function in the dog are unknown. We studied the uptake of beta-carotene by blood, corpus luteum, and uterine endometrium and the role of dietary beta-carotene in influencing ovarian steroid and uterine protein production during the estrous cycle in the dog. Mature female Beagle dogs (n = 56) were fed diets containing 0, 2, 20, or 50 mg of beta-carotene daily for approximately 6 wk before estrus detection. Blood was sampled at regular intervals from estrus through d 45 after ovulation (d 0 = ovulation), when laparotomy was performed. The ovaries were obtained for the isolation of corpus luteum. The uterus was flushed with phosphate-buffered saline and the endometrium obtained by scraping. Beta-carotene was not detectable in plasma, corpus luteum, or endometrium of unsupplemented dogs. However, beta-carotene and alpha-carotene in plasma, corpus luteum, and uterine endometrium increased in a dose-dependent manner. Alpha-carotene made up a high percentage of total carotenoids even though the alpha-carotene content in the dietary source was very low. Dogs fed 50 mg of beta-carotene had significantly higher concentrations of plasma progesterone between d 12 and 26 compared with unsupplemented dogs. Dietary beta-carotene did not influence plasma estradiol-17beta and total uterine proteins. Therefore, beta-carotene is absorbed into plasma, corpus luteum, and uterine endometrium of dogs. Furthermore, dietary beta-carotene increased plasma progesterone concentrations during the estrous cycle. It is possible that dietary beta-carotene may improve reproductive function in the canine.     

Presence of surfactant proteins in the uteri and placentae of pregnant mares

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.     

Case of Pregnancy in Two Cows with Unicorn Horn of the Uterus either by Artificial Insemination at Ipsilateral or Embryo Transfer at Contralateral Corpus Luteum in the Ovary

Two Holstein heifers and a cow were diagnosed with White Heifer Disease by ultrasonography. Case 1 was a 14 month-old heifer with aplasia of both sides of the uterine horn. In case 2, a primiparous cow and case 3, an 18 month-old heifer, both showed aplasia of the right uterine horn. Case 2 became pregnant by artificial insemination at ipsilateral ovulatory follicle and corpus luteum in the left ovary, while case 3 became pregnant by embryo transfer at 7 days after oestrus with contralateral corpus luteum in the right ovary.     

Myometrial electrical activity during pregnancy and parturition in the pygmy goat

To quantify the pattern of myometrial activity during gestation and parturition, one bipolar electrode was implanted on each uterine horn of four bilaterally pregnant pygmy goats between days 102 and 120 of gestation. After a recovery period, electromyographic recordings were made for at least six hours per day and continuously when parturition was judged to be imminent. During late gestation myometrial activity occurred as discrete episodes of myometrial electrical activity (EMEA) with a mean duration ranging from 6.2 +/- 2.07 to 8.3 +/- 1.60 minutes. The mean interval between two successive EMEAs ranged from 45.8 +/- 19.95 to 74.7 +/- 42.27 minutes. In three of the four goats these characteristics were not significantly different from the two uterine horns. Parturition was preceded by a prolonged period (eight to 12 hours) of myometrial quiescence. It was only from 19 to 15 hours before expulsion of the first kid that total duration of EMG activity increased. This finally resulted in the regular occurrence of bursts which occupied 25 to 30 per cent of the recording time. The results demonstrate that, as in the sheep, cow and pig, the myometrium is active during late pregnancy. It is postulated that luteolysis coincides with the prolonged period of myometrial quiescence which precedes the onset of the parturient pattern of uterine activity.     

Isolation and characterization of immunoglobulin binding proteins from Staphylococcus intermedius and Staphylococcus hyicus.

125-I-IgG binding activities were observed with 15 (17%) of 90 S. intermedius isolates from dogs and 39 (95%) of 41 S. hyicus isolates from pigs. Binding activities were not detected with S. hyicus isolates from cows. The IgG binding proteins of 2 S. intermedius, 2 S. hyicus, and protein A from S. aureus Cowan I were isolated from their cell surfaces. The proteins precipitated with IgG preparations from human, rabbit, pig, dog and horse, but not with IgG from cow, mouse and chicken. This indicated that these IgG binding proteins could be classified as type I receptors. In addition, the isolated proteins from all 3 staphylococcal species precipitated with polyclonal chicken anti-protein A antiserum. SDS-PAGE, Western blotting and gel isoelectric focussing of the proteins revealed numerous bands in the 42,000 D range and acid isoelectric points. The isoelectric point of the isolated proteins from both S. intermedius cultures was slightly more acidic than those from S. hyicus and S. aureus. The present results indicate a close functional and antigenic similarity, if not identity, between IgG binding proteins of S. intermedius and S. hyicus, and protein A of S. aureus.     

Secretory function of the ovine uterus: effects of gestation and steroid replacement therapy

In Exp. 1, changes in uterine fluid content of protein, calcium and prostaglandin F2 alpha (PGF) were examined between d 30 to 144 of gestation. Volume of uterine fluid (mean +/- SE) in the nongravid uterine horn of unilaterally pregnant ewes increased (P less than .05) between d 30 (8 +/- 1 ml) and d 144 (749 +/- 46 ml) of gestation. Protein concentration and total protein in uterine fluid also increased (P less than .05) between d 30 (2.3 +/- .4 mg/ml; 19 +/- 7 mg) and d 144 (23.5 +/- 3.3 mg/ml; 17.4 +/- 2.0 g). Total recoverable calcium (mg) in secretions increased from .1 mg (d 30) to over 1.4 g (d 144) due to day of gestation effects (P less than .05). Total PGF also increased (P less than .01) from 7 ng on d 30 to 15.7 g on d 144. In Exp. II, ovariectomized (OVX) ewes were treated with either corn oil, estrone (E), progesterone (P) or P+E (PE) for 30 d. Treatment with P or PE increased calcium concentration and content (P less than .01) in secretions, but differences in uterine fluid volumes and concentrations of protein and PGF were not significant. Treatment of OVX ewes with P stimulated (P less than .05) in vitro synthesis of secretory proteins by endometrium, whereas treatment with E enhanced release of unlabeled proteins (P less than .05). The major endometrial secretory proteins were identified in allantoic fluids from d 60 to term and were detected along the mesenchymal border of the chorioallantois using immunohistochemistry. Results from this study indicate that P may be a primary hormone regulating accumulation of fluid, protein, calcium and specific endometrial proteins in the uterine lumen during gestation, and that uterine milk proteins gain access to the fetal-placental unit.     

Appearance of the uterine specific proteins following induction of ovulation in prepubertal gilts.

The appearance of the uterine specific proteins following induction of ovulation in prepubertal gilts is described. 12 gilts each at 3, 4, and 5 months of age were allotted randomly to 1 of 2 treatment groups prior to induction of ovulation: 1) saline treated and 2) human chorionic gonadotropin (HCG) treated (400 IU daily from Days 12 through 16 of the induced cycle). Ovulation was induced with HCG following treatment with pregnant mare serum gonadotropin and the day of ovulation was designated as Day 1. All the gilts were laparotomized and uteri infused with phosphate-buffered saline on Day 16 to obtain uterine protein secretions. Plasma progesterone levels on Day 11 and observations made at laparotomy indicated that only 1 gilt 3 months of age failed to ovulate. The number of corpora lutea, plasma progesterone, total recoverable uterine protein, and uterine specific protein on Day 16 were markedly affected by the age of the gilt. These same characteristics, except uterine specific protein, were additionally affected by HCG treatment. Total recoverable uterine protein and uterine specific protein in saline and HCG-treated gilts at 3, 4, and 5 months of age were 6.3 and 1.5, 10.4 and 2.8; 38.8 and 15.2, 51.6 and 15.9; 20.4 and 7.7, 47.8 and 14.6 mg, respectively. Polyacrylamide slab gel electrophoresis showed that HCG-treated gilts at 3 months of age and both saline- and HCG-treated gilts at 4 and 5 months of age generally produced the purple basic protein and the complete profile of the low molecular weight acidic proteins during the induced cycle.     

Distribution patterns of mast cells in the uterus of pregnant Meishan pigs

The present study was performed to investigate the numerical distribution of mast cells (MCs) in the uteri of pregnant Meishan pigs to explore the functions of MCs in pig pregnancy. The uterine samples from pregnant (on days 15, 26 and 50 of gestation) pigs were obtained respectively and stained with toluidine blue. The results were as follows: MCs were constitutively located in the uterus of the Meishan pig, with the distribution varying with gestational stages. On days 15 and 26 of gestation, MCs were mainly distributed around the blood vessels and uterine glands within the endometrium. On day 50 of gestation, MCs were mostly distributed in the myometrium. These results indicated that uterine MCs possibly have versatile functions in pig pregnancy.