Increased expression of pro-inflammatory cytokines at the fetal-maternal interface in bovine pregnancies produced by cloning.

PROBLEM: A significant rate of spontaneous abortion is observed in cattle pregnancies produced by somatic cell nuclear transfer (SCNT). Major histocompatibility complex class I (MHC-I) proteins are abnormally expressed on the surface of trophoblast cells from SCNT conceptuses. METHOD OF STUDY: MHC-I homozygous compatible (n = 9), homozygous incompatible (n = 8), and heterozygous incompatible (n = 5) pregnancies were established by SCNT. Eight control pregnancies were established by artificial insemination. Uterine and trophoblast samples were collected on day 35 ±1 of pregnancy, the expression of immune-related genes was examined by qPCR, and the expression of trophoblast microRNAs was assessed by sequencing. RESULTS: Compared to the control group, trophoblast from MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL1A, IL2, IL10, IL12B, TBX21, and TNF, while GNLY expression was downregulated. The MHC-I homozygous incompatible treatment group expressed increased levels of IFNG, IL1A, and IL2 while the MHC-I homozygous compatible group did not differentially express any genes compared to the control group. In the endometrium, relative to the control group, MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL10, IL12B, and TNF, while GATA3 expression was downregulated. The MHC-I homozygous incompatible group expressed decreased amounts of CSF2 transcripts compared with the control group but did not have abnormal expression of any other immune-related genes. MHC-I incompatible pregnancies had 40 deregulated miRNAs compared to control pregnancies and 62 deregulated microRNAs compared to MHC-I compatible pregnancies. CONCLUSIONS: MHC-I compatibility between the dam and fetus prevented an exacerbated maternal immune response from being mounted against fetal antigens.

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy.

Trophoblast Major Histocompatibility Complex Class I Expression Is Associated with Immune-Mediated Rejection of Bovine Fetuses Produced by Cloning.

Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4(+) lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8(+), FOXP3(+), and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies.

Gross and histological description of the epidermal membrane found on normal neonatal piglets.

The structure and composition of fetal and placental tissues vary among different species of placental mammals. Some mammals, including camelids and pigs, possess an epidermal membrane during the fetal stage, called the epithelion, that is absent from other domestic mammals. Because neonatal piglets are a common animal model for many research techniques, it is important that researchers correctly identify this tissue and recognize its lack of pathological consequence when working with fetal and neonatal piglets. Here, the authors describe gross and histological examinations of the epithelion, comparing this tissue with other extra-fetal tissues and coatings.

Adult-onset lymphoplasmacytic orchitis in a Labrador retriever stud dog.

A formerly fertile 5-year-old 45-kg Labrador retriever was evaluated for azoospermia noted during routine semen collection for an artificial insemination. Over the past 3 years, the dog had sired 4 litters of anticipated size for the breed out of 5 breedings, the most recent a litter of 10 conceived and whelped 2 months previously. Physical examination findings were normal with the exception of bilaterally small and soft testes. An open excisional wedge biopsy of the right testis was performed under general anesthesia. Histopathology findings supported an immunologic, autoimmune pathogenesis that had resulted in infertility over the previous 4 months.

Histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (1990-2005).

OBJECTIVE: To determine the prevalence of various lesion types detected by histologic evaluation of uterine biopsy samples collected from subfertile bitches. DESIGN: Retrospective case series. ANIMALS: 399 sexually intact bitches. PROCEDURES: Results of histologic evaluation of canine uterine biopsy samples submitted by a single veterinary practice and clinical histories of dogs from which samples were obtained were reviewed. Clinical data including age, reason for biopsy, and histopathologic findings were recorded. The prevalence of specific lesions was determined, categorized by severity and age, and statistically analyzed. RESULTS: Endometritis (170/399 [42.6%] cases) and cystic endometrial changes, including cystic endometrial hyperplasia (133/399 [33.3%]) were the most prevalent lesions in the study population. Eighty-nine of 170 (52.4%) cases of endometritis were characterized as chronic with predominantly lymphocytic or lymphoplasmacytic inflammatory infiltrates, 51 (30.0%) included mixed inflammatory reactions, and 30 (17.6%) were characterized as having acute inflammation with neutrophils, eosinophils, or both. Fibrosis was common (101/399 [25.3%] cases). Eosinophilic endometritis was significantly associated with a history of fetal loss during the same breeding cycle. No significant difference was found in prevalence of lesions among age groups. CONCLUSIONS AND CLINICAL RELEVANCE: The high prevalence of endometritis in this population of dogs suggested that acute and chronic endometritis may be related to subfertility in bitches. The association of eosinophilic endometrial infiltrates with a history of fetal loss may be an important diagnostic finding in dogs with endometritis.

Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

Cerebrospinal fluid eosinophilia is a sensitive and specific test for the diagnosis of Parelaphostrongylus tenuis in camelids in the northeastern United States.

Aberrant migration of Parelaphostrongylus tenuis in camelids results in neurologic deficits, recumbency, and sometimes death. An antemortem diagnosis of P. tenuis in camelids is typically based upon the presence of characteristic asymmetric neurologic deficits, known exposure to white-tailed deer, cerebrospinal fluid (CSF) eosinophilia, and response to treatment. The diagnostic accuracy of CSF eosinophil percentage for the diagnosis of P. tenuis in camelids has not been critically examined. The objective of the current study was to determine the sensitivity (Se) and specificity (Sp) of CSF eosinophil percentage, CSF eosinophil concentration, total nucleated cell concentration, and protein concentration for the antemortem diagnosis of P. tenuis. Medical records of camelids admitted to Cornell University with clinical signs of neurologic disease, CSF analysis, and necropsy were examined from January 2000 through December 2009. Se and Sp were determined by receiver operating characteristic curves in camelids diagnosed with P. tenuis (n = 13) or other conditions (n = 24) based on postmortem examination. More than 17% of eosinophils in CSF had a Se of 85% and Sp of 92% for P. tenuis diagnosis (area under the curve [AUC]: 0.87; SE AUC: 0.07; P < 0.0001; 95% confidence interval [CI] AUC: 0.72-0.96), and >1.4 eosinophils/µl of CSF had a Se of 85% and Sp of 96% (AUC: 0.9; SE AUC: 0.06; P < 0.0001; 95% CI AUC: 0.76-0.97). Cerebrospinal fluid eosinophil percentage and concentration are sensitive and specific methods for diagnosing P. tenuis antemortem in camelids residing in regions endemic to white-tailed deer.

Findings in uterine biopsies obtained by laparotomy from bitches with unexplained infertility or pregnancy loss: an observational study.

Surgical uterine biopsies (SUBs) were obtained by laparotomy from 14 bitches with unexplained infertility whose pregnancy was not confirmed (NCP group) and from 7 bitches that had experienced unexplained pregnancy loss (PL). SUBs were obtained during the luteal phase of the cycle in 16 of 21 bitches. Overall, the biopsied uterine tissues presented obvious alterations in 17 of 21 cases. In the NCP group, 11 of 14 bitches had varying degrees of uterine lesions. In this group, six of nine bitches with macroscopically normal uteri presented histopathologic changes in the endometrium. All bitches with macroscopic modifications (five of 14) presented endometrial changes. Cytologic examination of the uterine fluid revealed eosinophilic amorphous material, erythrocytes, and a low number of degenerated leukocytes. On histopathologic examination, the most common lesion was fibrosis with degeneration of the endometrial glands (FDEGs six of 11), followed by endometritis (four of 11), cystic endometrial hyperplasia (CEH) (two of 11) and pseudoplacentational endometrial hyperplasia (PEH) (two of 11). Other lesions were adenomyosis, mucometra, and an endometrial polyp (one each of 11). Mixed lesions were present in four of the 11 bitches. In the PL group, macroscopic modifications consisted of intraluminal uterine dilatation (seven of seven) and presence of ovarian cysts (three of 7). Cytologic examination of the uterine content revealed high leukocyte counts. Histopathologic endometrial changes were found in six of seven bitches and included endometritis (three of seven), PEH (two of 6), pyometra (one of six), and CEH together with mucometra (one of six). Research of infectious agents was performed in 20 of 21 cases. Surprisingly, no bacteria or viruses were isolated from the uterine lumen in any of the cases. When the SUB was performed in diestrus, a medical treatment with aglepristone or with prostaglandins was established to avoid the risk of postoperative-induced pyometra. One case of complete uterine stenosis was the only side effect observed in the bitches that were not ovariohysterectomized after the collection of a biopsy (17 of 21). In conclusion, the most common findings in our biopsy specimens were FDEGs, endometritis, and hyperplastic remodeling of the endometrium (CEH and PEH). SUBs might provide useful information regarding the cause of infertility or PL. This technique allows visual inspection and palpation of the whole uterus and ovaries before the selection of a precise site for the biopsy. The size of the tissue sample collected can be large enough for an accurate diagnosis and samples for research of infectious agents can be obtained with no risk of contamination. Risk of side effects seems low when SUBs are performed according to this protocol, including postsurgery treatment with aglepristone or prostaglandins.

Diagnosis of Borrelia-associated uveitis in two horses.

Borrelia burgdorferi, the etiologic agent of Lyme disease is a tick born spirochetal infection. Clinical signs of Lyme borreliosis are uncommon in horses, but when present they are often vague and nonspecific. In horses, Lyme borreliosis has been implicated in musculoskeletal, neurological, reproductive, and ocular disorders, including uveitis, but definitive diagnosis can be challenging as the causative agent is rarely isolated and serologic tests can be unreliable and do not confirm active disease. Here, we report two cases of equine uveitis associated with B. burgdorferi based on the identification of spirochetes within ocular fluids and confirmed with PCR testing. The two cases illustrate some of the challenges encountered in the recognition and diagnosis of equine Lyme borreliosis. Although only one of many possible causes of equine uveitis, Lyme disease should be considered a differential diagnosis, especially in endemic areas. Given the possibility for false negative results of serum tests during uveitis associated with B. burgdorferi and the failure of such tests to confirm active infection, a combination of cytologic assessment, antibody, and/or PCR testing of ocular fluids may be worthwhile if the clinical suspicion for Lyme uveitis is high.

Chronic in vivo imaging in the mouse spinal cord using an implanted chamber.

Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics after a laser-induced spinal cord lesion and observed massive microglia infiltration within 1 d along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

Immunocontraception in Eastern gray squirrels (Sciurus carolinensis): morphologic changes in reproductive organs.

Eastern gray squirrels (EGS) (Sciurus carolinensis) damage trees through bark stripping or gnawing due to territorial marking or agonistic gnawing behavior in concert with higher densities. This study was conducted to determine the effects of a contraceptive vaccine on EGS and its reproductive organ histology. Free-ranging urban EGS were vaccinated with the immunocontraceptive GonaCon. All EGS were > or = 6 mo of age as determined by a combination of pelage characteristics and body weights. The vaccine was administered by injection at a dosage rate of 0.4 ml containing 400 microg of GnRH-mollusk protein conjugate i.m. in the thigh to 33 EGS (17 male [m], 16 female [f]) in trapping session 1 (TS1), 23 (14 m, 9 f) in trapping session 2 (TS2), and 11 (8 m, 3 f) in trapping session 3 (TS3). A sham injection containing 0.4 ml saline-AdjuVac was given as control to 22 EGS (16 m, 6 f) in TS1, 20 (12 m, 8 f) in TS2, and 8 (4 m, 4 f) in TS3. In the last trapping session (TS4), 35 EGS (16 treated, 19 control) were killed for necropsy to evaluate histologic changes in testes and ovaries. Treated EGS males had testicular, prostatic, and epididymal atrophy compared with control EGS males. The tubuli seminiferi and prostatic glandular lumen of treated EGS males were atrophic, and the epididymal lumen contained no sperm cells. No histologic changes were observed in treated EGS females; however, females likely were not collected when changes due to GonaCon would have been observed. There were no observable histologic differences in the pituitary gland of treated and control EGS. There were no statistically significant differences in either testosterone or progesterone concentrations between control and treated EGS. Although there were no serious side effects to the vaccine, six EGS developed injection site abscesses. GonaCon may be a potential tool for EGS population control.

A single base pair mutation encoding a premature stop codon in the MIS type II receptor is responsible for canine persistent Müllerian duct syndrome.

Müllerian inhibiting substance (MIS), a secreted glycoprotein in the transforming growth factor-beta family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In persistent Müllerian duct syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study, the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model could enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome.

Diffuse cutaneous mastocytosis in a newborn calf.

Previously reported cases of congenital cutaneous mast cell tumours in calves were characterized by multiple discrete nodules. The calf presented herein is, to the authors' knowledge, the first documented case of congenital diffuse cutaneous mastocytosis. The entire skin was thickened and wrinkled with particularly prominent skin folds on the head, ventrum, and legs. Histological examination of two haired skin specimens (eyelid, muzzle margin) revealed that the dermis was diffusely infiltrated by sheets of neoplastic mast cells.

Disorders of sexual differentiation in puppies and kittens: a diagnostic and clinical approach.

As in all domestic mammals, sexual differentiation in dogs and cats starts early in the embryonic period prenatally and continues into early postnatal life. The result of such a process is, however, not evident until after puberty,a time when the entire reproductive system undergoes significant changes. Normality of sexual differentiation is difficult to observe in neonates of small animals, with the only gender difference being a slightly longer anogenital distance in male (13-15 mm) versus female (7-8 mm)animals. Early diagnosis of deviations from normality can spare breeders the time and effort devoted to raising an animal that may turn out to be unsuitable for becoming part of the reproductive stock and may spare owners the concern for a pet whose health may be unnecessarily threatened by failing to remove a malformed reproductive system early in life. This article reviews the incidence, clinical and gross anatomic features,and diagnostic approaches that veterinarians can use to address inborn errors of the reproductive system of dogs and cats, highlighting those malformations that bear clinical relevance and may become manifest from birth until puberty.

Evidence for expression of both classical and non-classical major histocompatibility complex class I genes in bovine trophoblast cells.

PROBLEM: During the third trimester of pregnancy bovine trophoblast cells in the interplacentomal and arcade regions of the placenta express major histocompatibility complex class I (MHC-I) antigens. At parturition immunological recognition of MHC-I antigens appears to contribute to normal placental release. Therefore, we hypothesized that during late pregnancy bovine trophoblast cells express polymorphic, classical MHC-I antigens. METHOD OF STUDY: Cloning, microarray screening and sequencing of cDNA were used to study transcription of MHC-I genes in peripheral blood mononuclear cells (PBMC) and interplacentomal, trophoblast cells. Real-time reverse transcription, polymerase chain reaction (RT-PCR) was used to compare the abundance of MHC-I transcripts in PBMC and trophoblast cells. RESULTS: Screening of cloned MHC-I cDNA on MHC-I microarrays indicated that in PBMC 90-98% of MHC-I transcripts were encoded by classical MHC-I genes with the remainder encoded by non-classical MHC-I genes. In contrast, 21-66% of MHC-I transcripts from interplacentomal trophoblast cells were from classical genes and 34-79% were from non-classical genes. Transcripts from four non-classical MHC-I loci were identified by sequence analysis. Real-time RT-PCR indicated that the overall levels of MHC-I gene expression in PBMC and trophoblast were similar. CONCLUSION: Bovine interplacentomal trophoblast cells express both classical and non-classical MHC-I genes, but the relative level of expression varies considerably.