Estrogen-alpha and progesterone receptor expression in cystic endometrial hyperplasia and pyometra in the bitch

Estrogen-alpha receptor (ER) and progesterone receptor (PR) were examined immunohistochemically in uteri of normal bitches, in uteri of bitches with cystic endometrial hyperplasia-mucometra (CEH-M) and in uteri of bitches with endometritis-pyometra (E-P), under exogenous progesterone treatment.In the CEH-M group, the ER- and PR-scores of all uterine cell types were higher than the ER- and PR-scores of normal uteri, although these differences were not always statistically significant. The ER-scores of E-P group were significantly lower than the ER-scores of the normal uteri and CEH-M group. The PR-scores of the E-P group tended to be higher than the PR-scores of the normal uteri, except for the surface epithelium, although these differences were not statistically significant. Exogenous progesterone treated bitches with CEH-M or E-P showed reduced ER- and PR-scores in the different uterine cell types, compared with the corresponding nontreated CEH-M or E-P group.The differences in ER and PR expression between CEH-M and E-P suggest different factors in the pathogenesis of both entities. Although, these changes in ER and PR expression do not seem to be directly involved in the pathogenesis of CEH-M and E-P. It is suggested that for CEH-M and progestin induced CEH-M a hormone dependent pathway is responsible. For P, the trigger may be bacterial infection.     

A model for cystic endometrial hyperplasia/pyometra complex in the bitch

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.     

c-Jun N-terminal kinase regulates apoptosis in endometrial cancer cells

c-Jun N-terminal kinases (JNKs) are important regulators of cell proliferation and apoptosis that have been implicated in tumorigenesis. We investigated the role of JNKs in apoptotic responses in Ishikawa and HEC-50 cells, models of type I and type II endometrial cancer, respectively. Etoposide treatment or UV irradiation resulted in sustained activation of JNK, correlating with the induction of apoptosis. Inhibition of JNK, or MAP kinase kinase 4 (MKK4), selectively suppressed apoptotic responses in both Ishikawa and HEC-50 cells. Knockdown of protein kinase C δ (PKCδ) also attenuated apoptosis in endometrial cancer cells and inhibited the sustained, UV-mediated JNK activation in HEC-50, but not Ishikawa cells. Etoposide-induced JNK phosphorylation was unaffected by PKCδ knockdown, implying that JNK can regulate apoptosis by PKCδ-dependent and independent pathways, according to stimulus and cell type. Thus, expression and activity of JNK and PKCδ in endometrial cancer cells modulate apoptosis and sensitivity to chemotherapeutic agents and may function as tumor suppressors in the endometrium. Elaine M. Reno and James M. Haughian are first authors.     

Combination dopamine agonist and prostaglandin agonist treatment of cystic endometrial hyperplasia-pyometra complex in the bitch

Cystic endometrial hyperplasia-pyometra (CEH-P) complex is a progesterone-dependent disease that requires medical treatment in bitches intended for breeding. To test the efficacy and safety of a combined protocol and to assess the effect of age, stage of cycle, previous steroid hormone administration and parity on treatment, 29 bitches diagnosed with CEH-P complex were treated daily with cabergoline 5 microg/kg PO and cloprostenol 1 microg/kg SC for 7-14 days, along with supportive antibiotic and hydration therapies. Before treatment, and on Days 3, 7 and 14, all bitches were evaluated clinically and uterine horn diameter measured during trans-abdominal ultrasonography. Twenty-four of 29 bitches were cured by either Day 7 or 14. Nine bitches had mild digestive side effects. Clinical signs related to pyometra began to improve markedly as early as Day 2 of treatment. Uterine diameters decreased (P < 0.05) by Day 3 of treatment, and continued to gradually decrease, reaching normal size by Day 14. Relapses occurred in 6 of 29 cases. Pregnancy was achieved in one of the two young bitches bred after treatment. No significant relationships were found between success rate and age, stage of the estrous cycle, previous hormone administration or parity. Although no variables affecting treatment results could be identified, this combination of compounds was found to be an efficient and safe for treatment of CEH-P.     

Potential action of androstenedione on the proliferation and apoptosis of stromal endometrial cells

Background

Hyperandrogenic conditions have been associated with a high prevalence of endometrial pathologies related to cell survival. However, the action of androgens on proliferation and apoptosis in endometrial cells is poorly understood. Therefore, the aim of the present study was to evaluate the effect of androstenedione on cell proliferation, cell death and expression of estrogen receptor (ER) isoforms and proteins related to apoptosis in endometrial cells using two in vitro experimental approaches.

Methods

The endometrial tissue was obtained from 20 eumenorrheic women [28.7 (25 – 35) years] during the early secretory phase. We analyzed cell proliferation (immunohistochemistry of Ki-67 and spectrophotometric assay); apoptosis (DNA fragmentation (TUNEL) and Annexin V-FITC binding); ER-alpha, ER-beta bcl-2 and bax mRNA abundance (RT-PCR) in explants and isolated endometrial epithelial (EEC) and stromal cells (ESC) incubated with androstenedione 1 micro mol/l (A4) or A4 plus hydroxyflutamide 10 micro mol/l (F) for 24 h.

Results

In explants, A4 induced an increase of cell proliferation and a decrease on apoptosis in the stromal compartment (p < 0.05). In isolated ESC, proliferation augmented with A4 (p < 0.05), whereas, no significant modifications in the expression of ER-alpha, ER-beta bcl-2 and bax nor in the apoptotic index were observed. In EEC, A4 increase the ER-beta mRNA abundance (p < 0.05) and a decrease of the bcl-2/bax ratio (p < 0.05), without an increase in the apoptotic index. Hydroxyflutamide reverted the effect of androstenedione on the parameters described.

Conclusions

These results indicate that androstenedione may modulate cell survival, expression of ER-beta and proteins related to apoptosis, suggesting a potential mechanism that associates the effect of hyperandrogenemia on the endometrial tissue.

     

Possible role for insulin-like growth factor-I in the pathogenesis of cystic endometrial hyperplasia pyometra complex in the bitch

Cystic endometrial hyperplasia (CEH) is an important pathologic condition in the canine uterus and recognized as a common cause of illness and death in this species. The underlying cause and pathogenic mechanism responsible for this condition remains incompletely understood. Aberrant sex steroid hormone receptor expression in the uterus of dogs with CEH has been documented but not explained. In the dog there is an exceptionally high, progestin induced production of insulin-like growth factor-I (IGF-I) which is now generally accepted to be one of the most important growth factors with a high mitogenic effect on the uterus. Therefore, in this study the immunohistochemical staining intensity for IGF-I was compared among the uteri of 25 adult female dogs that had developed CEH and 14 healthy dogs in comparable stages of the estrus cycle. Specific staining for IGF-I was found in the cytoplasm epithelial cells and in smooth muscle cells of endometrium and myometrium. A marked increase in specific staining intensity for IGF-I was found in the surface epithelium, glandular epithelium and in the stroma of the uteri of dogs with CEH. The increase in IGF-I specific staining intensity was most prominent in the superficial endometrial stroma. Based on the known role of IGF-I in endometrial proliferation, it was concluded from the present study that high concentrations of IGF-I located in and around the epithelial cells of the endometrium in dogs with CEH, could play an important role in the development of CEH.     

Degeneration and death,by apoptosis and necrosis,of the pavement and chloride cells in the gills of the teleost Oreochromis mossambicus

Summary Degeneration and death of branchial epithelial cells were studied in an African cichlid fish. In both freshwater and seawater fish the superficially located pavement cells are sloughed off at the end of their lifecycle. This process is preceded by degeneration via a process of cytoplasmic shrinkage and condensation related to apoptotic (physiologically controlled) cell death. The chloride cells are pleomorphic, i.e., accessory, mature, and degenerating cells. Degeneration of chloride cells mainly occurs by apoptosis. Degenerating cells show shrinkage and densification of cytoplasm and nuclei, and swelling of the tubular system; these cells are then separated from the ambient water by pavement cells. They are finally phagocytosed and digested by macrophages. Apoptosis of chloride cells, but not of pavement cells, is greatly stimulated when the fish are in seawater; this reflects an increase in cellular turnover of the chloride cells. Accidental cell death (necrosis) of pavement cells or chloride cells is rarely observed in fully adapted freshwater and seawater fish. Its incidence increases in the first few days following transfer of fish from fresh water to seawater.     

The effects of endometrial scarification on uterine steroid receptors, bacterial flora and histological structure in the bitch.

Following laparotomy, the endometrium of six nulliparous Beagle bitches was scarified at the base of one uterine horn during early metoestrus, when the peripheral plasma P(4) concentration was >10 ng/ml; intrauterine swabs were taken at the same time for bacteriological culture. Twenty-one days later, a bilateral ovariohysterectomy was performed and segments of the scarified and non-scarified parts of the tubular genital tract removed; at the same time, swabs were taken from the uterine lumen. Tissue samples were collected and examined for histopathological structure, and the presence of nuclear oestrogen (ER) and progesterone (PR) receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score).All uterine swabs were sterile, and in three of the six bitches there were noticeable changes with distension of the uterine lumen with secretions and debris and distension of the endometrial gland ducts of the scarified uterine segment. There were no statistically significant differences in the H-scores of ER or PR between scarified and non-scarified segments, except for PR H-scores in the glandular epithelium where the values for the scarified were significantly higher than for the non-scarified endometrium (mean+/-S.E.M. is 129.9+/-22.8 versus 59.5+/-12.6; P<0.05). Thus, trauma can modify the structure of the endometrium and the characteristics of the PR. Whether changes in PR expression are involved in the pathogenesis of CEH/pyometra in the bitch could not be ascertained from this study.     

Mucometra, cystic endometrial hyperplasia, and pyometra in the bitch: advances in treatment and assessment of future reproductive success

Pyometra is a common reproductive disorder which affects nearly one fourth of all female dogs before they reach 10 y of age. An association between pyometra and the most common uterine disease of the bitch, cystic endometrial hyperplasia, has been established, as the latter allows commensal bacteria originating from the vagina to proliferate in the uterus at the end of estrus. The progressive degenerative process in the development of cystic endometrial hyperplasia is usually proposed as the initiating lesion for pyometra in bitches; this is mediated by progesterone and potentially aggravated by estrogens. However, a separate process caused by local uterine irritation to trophoblastic reaction and bacterial proliferation has been recently proposed as an alternate mechanism leading to the development of pyometra. Pyometra is clinically distinct in pathogenesis, signs, treatment and prognosis from postpartum metritis or mucometra. Treatment of pyometra has historically involved ovariohysterectomy, however, during the last 10 y, numerous effective treatments have been proposed to treat both open and closed cervix pyometra with good success and future fertility. Among the treatments available, the use of repeated low doses of prostaglandins alone or in association with either dopamine agonists or progesterone-receptor antagonists has been demonstrated to be a viable alternative for valuable breeding dogs.     

Cannabinoid-induced cell death in endometrial cancer cells: involvement of TRPV1 receptors in apoptosis

Among a variety of phytocannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most promising therapeutic compounds. Besides the well-known palliative effects in cancer patients, cannabinoids have been shown to inhibit in vitro growth of tumor cells. Likewise, the major endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), induce tumor cell death. The purpose of the present study was to characterize cannabinoid elements and evaluate the effect of cannabinoids in endometrial cancer cell viability. The presence of cannabinoid receptors, transient receptor potential vanilloid 1 (TRPV1), and endocannabinoid-metabolizing enzymes were determined by qRT-PCR and Western blot. We also examined the effects and the underlying mechanisms induced by eCBs and phytocannabinoids in endometrial cancer cell viability. Besides TRPV1, both EC cell lines express all the constituents of the endocannabinoid system. We observed that at concentrations higher than 5 μM, eCBs and CBD induced a significant reduction in cell viability in both Ishikawa and Hec50co cells, whereas THC did not cause any effect. In Ishikawa cells, contrary to Hec50co, treatment with AEA and CBD resulted in an increase in the levels of activated caspase ?3/?7, in cleaved PARP, and in reactive oxygen species generation, confirming that the reduction in cell viability observed in the MTT assay was caused by the activation of the apoptotic pathway. Finally, these effects were dependent on TRPV1 activation and intracellular calcium levels. These data indicate that cannabinoids modulate endometrial cancer cell death. Selective targeting of TPRV1 by AEA, CBD, or other stable analogues may be an attractive research area for the treatment of estrogen-dependent endometrial carcinoma. Our data further support the evaluation of CBD and CBD-rich extracts for the potential treatment of endometrial cancer, particularly, that has become non-responsive to common therapies.     

Pregnancy management in the bitch

Pregnancy management to optimize maternal and neonatal health begins with breeding management and the selection of normal, healthy brood stock in ideal body condition. After breeding, a commercial diet appropriate for reproduction and lactation should be fed. Typically these contain 29–32% protein of animal source, at least 18% fat, 20–30% carbohydrate, and essential vitamins, minerals and fatty acids. Pregnancy is confirmed approximately 25 d after breeding. A “maternity ward” and whelping box should be provided. Steady increases in caloric intake and body weight are expected as pregnancy progresses. Weight loss should not occur. Throughout pregnancy, changes in the bitch’s attitude, activity, appetite, body weight, and physical findings should be monitored by the owner. If appetite and body weight do not continue to increase, or if any signs of illness develop, maternal health should be assessed with a complete physical examination and a CBC, biochemical profile, and free-catch urinalysis. Fetal health should be assessed with ultrasonography. Maternal or fetal abnormalities will put the pregnancy at risk. Impending parturition and the progress of labor and delivery can be monitored by assessing rectal temperature, serum concentrations of progesterone, and/or uterine and fetal monitors. This article reviews the physiology of canine pregnancy and parturition, and typical schemes used to manage normal canine pregnancy to optimize maternal and puppy health.     

Pregnancy management in the bitch

Pregnancy management to optimize maternal and neonatal health begins with breeding management and the selection of normal, healthy brood stock in ideal body condition. After breeding, a commercial diet appropriate for reproduction and lactation should be fed. Typically these contain 29-32% protein of animal source, at least 18% fat, 20-30% carbohydrate, and essential vitamins, minerals and fatty acids. Pregnancy is confirmed approximately 25 d after breeding. A "maternity ward" and whelping box should be provided. Steady increases in caloric intake and body weight are expected as pregnancy progresses. Weight loss should not occur. Throughout pregnancy, changes in the bitch's attitude, activity, appetite, body weight, and physical findings should be monitored by the owner. If appetite and body weight do not continue to increase, or if any signs of illness develop, maternal health should be assessed with a complete physical examination and a CBC, biochemical profile, and free-catch urinalysis. Fetal health should be assessed with ultrasonography. Maternal or fetal abnormalities will put the pregnancy at risk. Impending parturition and the progress of labor and delivery can be monitored by assessing rectal temperature, serum concentrations of progesterone, and/or uterine and fetal monitors. This article reviews the physiology of canine pregnancy and parturition, and typical schemes used to manage normal canine pregnancy to optimize maternal and puppy health.     

High-risk pregnancy and hypoluteoidism in the bitch

High-risk pregnancies are those in which the prevalence of maternal, fetal and/or perinatal morbidity or mortality is likely to be higher than that of the general obstetrical population. Some maternal characteristics associated with risk to maternal, fetal and/or perinatal health are readily identifiable prior to conception, such as advanced maternal age, brachycephalic breed, or a previous history of pregnancy loss. Others, such as gestational diabetes or a singleton litter, are recognized after conception. Early recognition of the problem (i.e. the risk), anticipation of the potential sequelae, and development of an aggressive management scheme are essential for a successful outcome of a high-risk pregnancy. A previous history of pregnancy loss is a high-risk factor for recurrence during subsequent pregnancies. Infection is a common cause. In some instances, recurrent pregnancy loss is associated with low serum concentrations of progesterone. Although the mechanism(s) by which this occurs is not fully understood, the situation has been called hypoluteoidism. Whatever the cause of the risks to pregnancy, the goals of managing high-risk pregnancies are to optimize maternal, fetal and perinatal health, so as to maintain maternal health throughout pregnancy and lactation and maximize the number of healthy pups surviving to weaning age.     

Icaritin causes sustained ERK1/2 activation and induces apoptosis in human endometrial cancer cells

Icaritin, a compound from Epimedium Genus, has selective estrogen receptor (ER) modulating activities, and possess anti-tumor activity. Here, we examined icaritin effect on cell growth of human endometrial cancer Hec1A cells and found that icaritin potently inhibited proliferation of Hec1A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinD1 and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, icaritin induced sustained phosphorylation of extracellular signal-regulated kinase1/2 (the MAPK/ ERK1/2) in Hec1A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by icaritin and abolished the icaritin-induced growth inhibition and apoptosis. Our results demonstrated that icaritin induced sustained ERK 1/2 activation and inhibited growth of endometrial cancer Hec1A cells, and provided a rational for preclinical and clinical evaluation of icaritin for endometrial cancer therapy.     

High-risk pregnancy and hypoluteoidism in the bitch

High-risk pregnancies are those in which the prevalence of maternal, fetal and/or perinatal morbidity or mortality is likely to be higher than that of the general obstetrical population. Some maternal characteristics associated with risk to maternal, fetal and/or perinatal health are readily identifiable prior to conception, such as advanced maternal age, brachycephalic breed, or a previous history of pregnancy loss. Others, such as gestational diabetes or a singleton litter, are recognized after conception. Early recognition of the problem (i.e. the risk), anticipation of the potential sequelae, and development of an aggressive management scheme are essential for a successful outcome of a high-risk pregnancy. A previous history of pregnancy loss is a high-risk factor for recurrence during subsequent pregnancies. Infection is a common cause. In some instances, recurrent pregnancy loss is associated with low serum concentrations of progesterone. Although the mechanism(s) by which this occurs is not fully understood, the situation has been called hypoluteoidism. Whatever the cause of the risks to pregnancy, the goals of managing high-risk pregnancies are to optimize maternal, fetal and perinatal health, so as to maintain maternal health throughout pregnancy and lactation and maximize the number of healthy pups surviving to weaning age.     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.     

Laparoscopic intrauterine insemination in the bitch

A technique for laparoscopic intrauterine insemination in bitches is described. During natural estrus, 5 beagle bitches were inseminated and S others were naturally mated (control group) twice at a 48-h interval on Days 3 and S (n = 4) or Days 4 and 6 (n = 6) after the increase in plasma progesterone considered to be indicative of the day of the preovulatory LH peak. All the inseminations were with fresh semen and under general anesthesia. The technique involved the introductions of 1) a Verres needle to insufflate the abdominal cavity by direct punction on the middle line 1 cm over the umbilicus, 2) a laparoscope to visualize the abdominal cavity by a 1 cm puncture on the middle line 1 cm under the umbilicus, 3) a forceps used to manipulate the uterus by a 0.5 cm puncture at 2 to 3 cm lateral to the mammary glands, and 4) an 18-g catheter used to puncture the uterus on the middle line between the 3rd and 5th mammary gland. The uterine body was grasped by the forceps and elevated against the ventral abdominal wall. The 18-g catheter was then inserted through the abdominal wall directly into the uterine lumen, and 1.0 ml of fresh semen containing 250 to 480 x 10(6) spermatozoa/ml was injected. The inseminations resulted in pregnancies in all animals. Litter size was similar in the artificially inseminated and naturally mated bitches (5 +/- 1.8 and 4.8 +/- 1.6 pups per litter, respectively). Bitches in the artificially inseminated group delivered at 65.2 +/- 0.8 d and in the natural mated group at 65.4 +/- 0.5 d after the LH peak. In conclusion, this paper gives the first results of intrauterine laparoscopic insemination in bitches, indicating interesting perspectives for this technique in dog's reproduction.     

Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture

Cryopreservation causes a significant proportion of bovine oocytes to undergo degeneration during subsequent culture. We investigated the degeneration mechanism of cryopreserved oocytes. In vitro matured bovine oocytes were vitrified by the open-pulled straw (OPS) method. In each replicate, a group of oocytes were randomly taken after warming to determine oocyte survival by both morphological evaluation and propidium iodide vital staining. The remainders were evaluated by morphological criterion. Morphologically intact oocytes were co-incubated with frozen-thawed spermatozoa for subsequent development. In situ examination of DNA breaks in oocytes and embryos was conducted using a Fluorescein-FragEL DNA fragmentation detection kit. A caspase-3 detection kit was used to detect caspase-3 activity in oocytes and embryos. Most of the oocytes survived cooling and warming processes as assessed by both morphological evaluation and vital stain. During subsequent culture, some degenerating oocytes displayed observable apoptotic morphology, such as cytoplasmic condensation, cytoplasmic fragmentation, and formation of apoptotic bodies. Biochemical markers of apoptosis, such as apoptotic DNA fragmentation and activation of caspases, were detected not only in oocytes having typical apoptotic morphology, but also in oocytes without observable apoptotic morphology. In embryos, positive signals for both biochemical markers were detected in blastomeres. This experiment suggests that cryopreserved bovine oocytes degenerate via apoptosis during subsequent culture.