Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat.

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

Predicting nuclear maturation speed of oocytes from Japanese Black beef heifers through non-invasive observations during IVM: An attempt using machine learning algorithms.

Nuclear maturation is an essential process in which oocytes acquire the competence to develop further. However, the time required for nuclear maturation during IVM varies among oocytes. Therefore, predicting nuclear maturation speed (NMS) could help identify the optimal timing for IVF and maximize the developmental competence of each oocyte. This study aimed to establish machine learning-based prediction models for NMS using non-invasive indicators during the individual IVM of Japanese Black (JB) beef heifer oocytes. We collected ovaries from abattoirs and aspirated cumulus-oocyte complexes (COCs) from follicles with diameters ranging between 2 and 8 mm. The COCs were matured individually for 18 h, and photographs of each COC were taken at the beginning and every 3 h from 12 h to the end of maturation. After IVM culture, we denuded COCs and stained oocytes to confirm the progression of meiosis. Only oocytes that reached the metaphase II (MII) stage were considered to have a fast NMS. Morphological features, including COC area, cumulus expansion ratio, expansion rate per hour, and expansion pattern, were extracted from the recorded photos and applied to develop prediction models for NMS using machine learning algorithms. The MII rates of oocytes with fast- and slow-predicted NMS differed when the decision tree (DT) and random forest (RF) models were employed (P < 0.05). To evaluate the relationship between predicted NMS by DT and RF models and fertilization dynamics during individual IVF, sperm penetration and pronuclear formation were evaluated at 3, 6, 9, and 12 h after IVF start, following 24 h of IVM. The association between predicted NMS and embryo development was investigated by performing IVC for seven days using microwell culture dishes after 24 h of IVM and 6 h of IVF. Predicted NMS did not show a significant association with fertilization dynamics. However, oocytes with fast-predicted NMS by the RF model exhibited a tendency towards a higher cleavage rate 48 h after IVF start (P = 0.08); no other relationship was found between predicted NMS and embryo development. These findings demonstrate the feasibility of using non-invasive indicators during IVM to develop prediction models for NMS of JB beef heifer oocytes. Although the effect of predicted NMS on embryo development remains unclear, customized treatments based on NMS predictions have the potential to improve the efficiency of in vitro embryo production following individual IVM culture.

Insulin-like peptide 3 in domestic animals with normal and abnormal reproductive functions, in comparison to rodents and humans.

Background: Insulin-like peptide 3 (INSL3) is a circulating hormone secreted from only testis and ovaries in mammals. Findings on INSL3 have been gathered from subjects with normal and abnormal reproductive statuses, especially rodents and humans. However, little to no review articles focusing on INSL3 in domestic animals exist. Methods: The author reviewed the past and recent literature regarding the structure, expression, roles of INSL3 in the reproductive organs, and its circulation under normal and aberrant reproductive conditions in domestic animals in comparison with rodents and humans. Main findings: As with humans and rodents, blood INSL3 concentrations rise around puberty in normal male domestic animals and are associated with testicular size. INSL3 levels are acutely upregulated by luteinizing hormone (LH), and the increase is smaller than that of testosterone in male ruminants, whereas the acute regulation of INSL3 by LH does not occur in human men. Dogs with cryptorchidism and bulls with abnormal semen have lowered INSL3 levels. Conclusion: The findings regarding INSL3 secretions in male domestic animals with normal and aberrant reproductive functions illustrate similar or dissimilar points to humans and rodents. Data on blood INSL3 levels in normal and abnormal female domestic species are still limited and require further investigation.

Quantitative analyses of insulin-like peptide 3 and sex steroid hormones in dominant follicles and corpora lutea during the estrus cycle and in follicular cysts in beef heifers.

Insulin-like peptide 3 (INSL3) and sex steroids were measured in bovine dominant follicles and corpora lutea during the estrus cycle and in follicular cysts. Paired ovaries from beef heifers (n = 47) were classified, by their morphological features, either into four stages of the estrus cycle (Day 1 = day of ovulation, Day 20 = day of estrus) as Stage I (Days 1-4; n = 8), Stage II (Days 5-10; n = 10), Stage III (Days 11-17; n = 8), and Stage IV (Days 18-20; n = 11) or follicular cystic (n = 10). Cysts (n = 15) were subdivided into estrogen-active (n = 7) and estrogen-inactive (n = 8) cysts. INSL3, testosterone, and estradiol-17ß concentrations in the dominant follicular fluid of Stage IV were higher than those in Stages II and III (P < 0.05). INSL3 concentrations in the cystic fluid were similar to those in dominant follicles at Stage IV, whereas testosterone and estradiol-17ß concentrations were lower in cysts (P < 0.05). INSL3 content per estrogen-inactive cyst was higher than that of Stage IV (P < 0.05). INSL3 and progesterone concentrations in luteal tissue and contents per corpus luteum were higher in Stages II and III (P < 0.05). In conclusion, INSL3 secretion in bovine dominant follicles increased with maturation. Follicular cysts may retain the production of INSL3 during their formation but tend to lose the capacity for testosterone secretion. Estrogen-inactive cysts subjected to advanced atresia may accumulate more INSL3. INSL3 production in bovine corpora lutea is enhanced during maturation.

Anti-Müllerian hormone, testosterone, and insulin-like peptide 3 as biomarkers of Sertoli and Leydig cell function during deslorelin-induced testicular downregulation in the dog.

The role of anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3) in male infertility is not fully understood. We used the downregulated testis as a model of gonadotropin-dependent infertility. Serum testosterone and AMH concentrations were studied in five adult male Beagles implanted (day 0) with 4.7 mg deslorelin (Suprelorin®, Virbac) (DES group). Testicular expression of LH receptor (LHR) and androgen receptor (AR), AMH, type 2 AMH receptor (AMHR2), INSL3 and its receptor (RXFP2) was evaluated 112 days (16 weeks) after deslorelin treatment by qPCR and immunohistochemistry, and compared to untreated adult (CON, n = 6) and prepubertal (PRE, n = 8) dogs. Serum testosterone concentration decreased significantly by the onset of aspermia on study day 14 (four dogs) or day 21 (one dog), and was baseline on day 105 (week 15). In contrast, serum AMH started to increase only after the onset of aspermia and reached the maximum detectable concentration of the assay by day 49-105 in individual dogs. Testicular LHR gene expression in DES was lower than in CON and PRE (P < 0.0001), while AR gene expression in DES was similar to CON and significantly higher than PRE (P < 0.0001). Testicular AMH expression in DES was intermediate compared to the lowest mRNA levels found in CON and the highest in PRE (P ≤ 0.006). AMHR2 gene expression was similar between groups. AMH protein was detected in Sertoli cells only, while AMHR2 immunoreactivity was principally detected in Leydig cells which appeared to be increased in DES. INSL3 and RXFP2 gene expression was significantly downregulated in the DES testis along with noticeably weak Leydig cell immunosignals compared to CON. In conclusion, deslorelin treatment caused testicular LH insensitivity without affecting androgen sensitivity, and de-differentiation of Sertoli and Leydig cells. In DES, upregulation of the AMH-AMHR2 feed-back loop and downregulation of the INSL3-RXFP2 feed-forward loop are paracrine-autocrine mechanisms that may additionally regulate testosterone production independent of gonadotropins. Our results support AMH and INSL3 as unique biomarkers and paracrine-autocrine regulators of testis function involved in the intimate interplay between Sertoli and Leydig cells.

Inhibitory effects of long-term repeated treatments of a sustainable GnRH antagonist, degarelix acetate, on caprine testicular functions.

We investigated the effects of long-term repeated treatments with a sustainable gonadotropin-releasing hormone (GnRH) antagonist, degarelix acetate, on testicular hormonal secretion, size, ultrasound images, histology and spermatogenesis in goats to assess its efficacy as a chemical castration method. Male Shiba goats (3-6 months of age) were treated subcutaneously with degarelix acetate every 4 weeks for 24 weeks. Plasma testosterone and insulin-like peptide 3 concentrations decreased (P < 0.05) within 2 days after the first treatment and remained low until 29 weeks (P < 0.05). Scrotal circumference and testicular pixel intensity were lower from 2-6 months and from 1-6 months, respectively, compared to the pretreatment values (P < 0.05). The testis and epididymis weights were lower at 24 weeks compared to those in untreated goats (P < 0.05). There were no sperm in the seminiferous tubules of testicular tissue sections or in homogenates of the epididymis at 24 weeks. These results suggest that repeated treatment with degarelix acetate is an effective chemical castration method for goats.

Serum concentrations and testicular expressions of insulin-like peptide 3 and Anti-Müllerian hormone in normal and cryptorchid male horses.

Insulin-like peptide 3 (INSL3) is an important hormone for testicular descent during embryonic development and a factor for assessing functional status of Leydig cells of testes, but there is limited number of equine studies. Anti-Müllerian hormone (AMH) is a useful diagnostic marker for cryptorchidism in horses. This study aimed to compare serum concentrations and testicular expression intensity of INSL3 and AMH in intact and cryptorchid male horses. Serum INSL3 concentrations in intact (n = 9; mean ± SEM, 19.9 ± 5.9 ng/mL) and noncastrated unilateral cryptorchid (UC) male horses (n = 16; mean ± SEM, 16.8 ± 4.1 ng/mL) were higher compared with hemicastrated unilateral cryptorchid (HCUC) male horses (n = 9; mean ± SEM, 3.8 ± 0.7 ng/mL) (P < 0.05). And serum INSL3 in bilateral cryptorchid (BC) male horses (n = 4; 1.9 ± 0.4; mean ± SEM, ng/mL) were lower compared with intact male horses (P < 0.05). Serum AMH concentrations in BC male horses (n = 3; mean ± SEM, 30.6 ± 4.8 ng/mL) were higher compared with intact male horses (n = 5; mean ± SEM, 12.2 ± 3.9 ng/mL) (P < 0.05). Immunostaining of scrotal and cryptorchid testis showed that Sertoli cells were positive for AMH, and Leydig cells were positive for INSL3. Staining intensity of AMH was higher in cryptorchid testis than in scrotal testis (P < 0.05). Furthermore, AMH expression intensity was higher in abdominal testis than in inguinal testis (P < 0.05). Immunostaining intensity of INSL3 in the testis was positively correlated with serum INSL3 (r, 0.7; P < 0.01), seminiferous tubule area (r, 0.727; P < 0.01), and Johnsen score for spermatogenesis (r, 0.604; P < 0.05), whereas immunostaining intensity of AMH in the testis was negatively correlated with seminiferous tubule area (r, -0.814; P < 0.01) and Johnsen score for spermatogenesis (r, -0.807; P < 0.01). Our findings suggested that AMH is a good biomarker for diagnosing cryptorchidism in male horses, in addition to INSL3 values to assess the testis of intact and cryptorchid male horses.

Age related and seasonal changes of plasma concentrations of insulin-like peptide 3 and testosterone from birth to early-puberty in Thoroughbred male horses.

The peripheral blood concentrations of insulin-like peptide 3 (INSL3) have been detected in many mammalian species, but the level of INSL3 in horse remains unknown. The objectives were to develop a time-resolved fluorescence immunoassay (TRFIA) to detect INSL3 concentrations from horse blood as well as to determine the age-related and seasonal changes of plasma concentrations of INSL3 and testosterone from birth to early-puberty in Thoroughbred male horse (n = 11). Monthly blood sample and measurement of body weight, height, chest and cannon bone size were done from birth until 16 mo. The TRFIA and EIA were used to measure plasma concentrations of INSL3 and testosterone, respectively. An increase in mean body weight, height, chest and cannon bone size was observed throughout the study. The monthly blood sampling revealed an increase in mean plasma INSL3 concentrations up to 2 mo, followed by a decreasing and increasing pattern until the end of experiment at 16 mo. A high testosterone level was detected at birth followed by a sharp decrease to basal level within 1 mo, maintained low level up to10 mo before a gradual rise until 16 mo. In case of seasonality, there was no difference in mean plasma INSL3 concentrations between breeding (March to September) and non-breeding (October to February) seasons, whereas a higher (P < 0.001) mean plasma testosterone concentrations in the second breeding season compared to non-breeding season was observed. In age categorized group, an increase (P < 0.01) in mean plasma INSL3 concentrations was noticed at pre-puberty (1-12 mo) and early-puberty (13-16 mo) compared to birth, but a lower (P < 0.001) mean plasma testosterone concentrations was observed at pre-puberty compared to birth and early-puberty. In conclusion, a TRFIA was developed to measure INSL3 levels in horse. An increase in plasma concentrations of INSL3 and testosterone were observed with the advancement of age, whereas for testosterone a very lower level was detected at the non-breeding season than in the second breeding season after birth in Thoroughbred male horse. The INSL3 secretions seemed independent of seasonal influence, at least before puberty.

Feline embryo development in commercially available human media supplemented with fetal bovine serum.

Feline embryo development was examined for 7 days after fertilization using commercially available human media supplemented with 0.3% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS). Cumulus-oocyte complexes were categorized as Grades 1, 2, and 3 according to morphology. Only-One Medium (OM) was used for in vitro culture (IVC) in OM + BSA, OM + FBS, and OM + BSA/FBS, with BSA supplementation for the first 2 days and FBS for the subsequent 5 days. Embryos cultured in Early Culture Medium (1-2 days) and Blastocyst Medium (3-7 days) were defined as EB + BSA and EB + BSA/FBS. The developmental rate until the blastocyst stage of Grade 1 and 2 oocytes cultured in OM + BSA/FBS was higher than for the other groups and was significantly higher than for the OM + BSA and EB + BSA groups (P<0.01). Grade 3 oocytes cultured in OM + BSA/FBS also showed the greatest proportion of blastocyst formation. However, FBS supplementation throughout the IVC period reduced blastocyst number. The percentage of 2 pronuclei after fertilization as well as blastocyst cell number were significantly higher in Grade 1 and 2 than Grade 3 oocytes when cultured in OM + BSA/FBS (P<0.05). These results indicate that commercially available OM supplemented with BSA for the first 2 days of culture and FBS for the subsequent 5 days is suitable for feline embryo development until the blastocyst stage.

Production of Japanese Black calves by the transfer of embryos developed from in vitro-fertilized oocytes derived by ovum pick up and matured in culture with the mitogen-activated protein kinase kinase inhibitor U0126.

This study investigated whether treatment with the mitogen-activated protein kinase kinase inhibitor U0126 during in vitro maturation (IVM), which has previously been reported to improve oocyte developmental competence, is practical for use in calf production using ovum pick up (OPU)-derived oocytes. Two Japanese Black cows were repeatedly and simultaneously treated to stimulate follicular growth and were prepared for OPU. Cumulus-oocyte complexes (COCs) were collected from one cow using a collection medium containing 5 µM U0126 and were cultured in medium supplemented with the same concentration of U0126 for the first 2 hr of IVM; COCs from the other cow were used as controls without U0126 treatment. The cows were exchanged between the two groups at every sequential OPU (n=8). The number of oocytes developing to blastocysts in the U0126-treated group (39.1%, 34/87) was significantly higher than that in the control group (22.1%, 19/86). Eight blastocysts produced with U0126 treatment were transferred to recipients, and four normal calves were obtained. The results indicate that embryos develop efficiently from OPU-derived oocytes treated with U0126, and that these embryos may be of practical use in calf production.

LH and testosterone secretions in response to GnRH challenge in pubertal Japanese Black beef bulls with normal and abnormal semen.

Plasma luteinizing hormone (LH) and testosterone concentrations were examined in Japanese Black beef bulls with normal and abnormal semen in response to gonadotropin releasing hormone (GnRH) challenge at the start (10 months) and completion (20 months) of puberty. Bulls with normal semen had higher testosterone concentrations after GnRH treatment at 20 months than they did at 10 months, while LH concentrations did not differ between the two age groups. LH and testosterone concentrations were not different between bulls with normal and abnormal semen at 20 months. Thus, testosterone secretions in response to the GnRH challenge were higher for bulls with normal semen at pubertal completion compared to bulls at the start of puberty, but responsiveness of LH to GnRH and of testosterone to the LH increment was not altered in bulls with abnormal semen.

Rescue of the fetal damage associated with high intrauterine pressure by 17ß-estradiol injection in ovariectomized progesterone-treated pregnant mice.

The present study examined the effects of progesterone (P) and 17ß-estradiol (E2) on fetal damage and intrauterine pressure in ovariectomized pregnant mice. The mice were ovariectomized on gestational day (GD) 9 (copulation plug = GD 0), and daily subcutaneous injection of various doses of P (2, 3 or 4 mg) or 4 mg P plus E2 (0.05 or 0.1 µg) was given thereafter. Although P alone increased percentage of normal fetuses on GD 17 dose-dependently, fetal injury with edematous hematomata on their extremities was frequently observed. In the group treated with 4 mg P, the injured fetus was found at the highest percentage (18%) and intrauterine pressure was significantly higher than that in intact pregnant mice (controls). No injured fetus on GD 17 was found by the treatment with 4 mg P plus 0.05 or 0.1 µg E2, and the treatments decreased the intrauterine pressure to the level of controls. Percentage of normal fetuses in the ovariectomized mice treated with 4 mg P plus 0.05 µg E2 was similar to that of controls, while that in the ovariectomized mice treated with 4 mg P plus 0.1 µg E2 markedly decreased. The results suggest that estrogen decreases intrauterine pressure to defend fetal damage in ovariectomized P-treated mice, and a high estrogen level interrupted pregnancy while keeping this estrogen action.

EFFECTS OF GONADOTROPIN-RELEASING HORMONE ANTAGONIST DEGARELIX ON MUSTH AND SERUM TESTOSTERONE CONCENTRATIONS IN ASIAN ELEPHANTS ( ELEPHAS MAXIMUS).

Two male Asian elephants (bulls 1 and 2) in musth were subcutaneously injected with a long-acting gonadotropin-releasing hormone (GnRH) antagonist, degarelix acetate (240 µg/kg; total dose of 960 mg). Musth behavior (MB) and temporal gland secretions (TGS) were monitored and serum testosterone concentrations were determined. In bull 1, MB and TGS ceased on day 1 and reappeared 5.5 mo after the treatment (day 0). During the subsequent musth cycle, MB and TGS ceased on day 1 and did not appear for 4 mo. In bull 2, MB and TGS ceased at day 7 after the treatment. Musth behavior and TGS recurred on Day 11 and continued for 1 wk, then disappeared for 8 mo. Serum testosterone concentrations decreased ( P < 0.05) in all occasions from day 0 (29.8 ± 15.8 ng/ml; mean ± SEM) to day 1 (2.2 ± 1.1 ng/ml), suggesting a sudden drop in circulating testosterone in musth elephants after the GnRH-antagonist treatment.

Relationships of plasma insulin-like peptide 3, testosterone, inhibin, and insulin-like growth factor-I concentrations with scrotal circumference and testicular weight in Japanese Black beef bull calves.

This study was conducted to clarify the relationships of plasma concentrations of insulin-like peptide 3 (INSL3), testosterone, inhibin, and insulin-like growth factor-I (IGF-I) with scrotal circumference and testicular weight in Japanese Black beef bull calves (n = 20), from birth to pre-puberty. Monthly blood sampling (0 to 7 months) and scrotal circumference measurements (0 to 7 months) were performed. Testicular weight was recorded immediately after castration at 7 months. Plasma INSL3, testosterone, inhibin, and IGF-I concentrations were measured either by enzyme immunoassay or time-resolved fluorescence immunoassay. The correlation coefficients of these hormonal concentrations with scrotal circumference were significant (P < 0.0001) and it was higher for INSL3 (r = 0.647) than for testosterone (r = 0.597), IGF-I (r = 0.400), and inhibin (r = -0.453). Calves with heavier testes (> 60 g) at castration (7 months) had higher (P < 0.05) plasma INSL3 (from 3 to 7 months) and inhibin (from 1 to 4 months) concentrations than those with lighter testes (< 60 g). The calves with heavier testes at castration had larger (P < 0.05) scrotal circumference than those with lighter testes from 3 to 7 months. In conclusion, blood INSL3 concentrations may be the best functional indicator among the hormones analyzed for determining total testicular volume during pre-puberty in bull calves. In addition, inhibin and INSL3 concentrations in early calfhood may be functional predictors for testicular weight at pre-puberty.

Efficacy of mechanical micro-vibration in the development of bovine embryos during in vitro maturation and culture.

It is currently unclear how mechanical micro-vibration affects the in vitro culture of embryos in Japanese Black cow. In the experimental groups, immature oocytes and fertilized embryos were cultured using the micro-vibration culture system with the vibration set for 5 sec at intervals of 60 min and frequency of 20, 40 or 80 Hz, respectively, during in vitro maturation and in vitro development. Compared with the control group, the rate of blastocyst development significantly increased in the 40 Hz group. In addition, the number of blastocyst cells reduced significantly in the 80 Hz group. In conclusion, the development of blastocysts in cows is facilitated by providing moderate mechanical micro-vibration to immature oocytes and embryos during the in vitro maturation and in vitro development.

Generation of Canine Induced Extraembryonic Endoderm-Like Cell Line That Forms Both Extraembryonic and Embryonic Endoderm Derivatives.

Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4. Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.

Exposure to Zearalenone During Early Pregnancy Causes Estrogenic Multitoxic Effects in Mice.

Although zearalenone (ZEN; Sigma Chemicals, St Louis, Missouri) is a well-known mycotoxin with estrogenic activity, the toxic effects of ZEN during pregnancy are unknown. This study compared the effects of daily subcutaneous injections of ZEN (2, 4, or 8 mg/kg) with those of 17ß-estradiol (E2; [Sigma Chemicals] 0.8, 1.6, or 3.2 µg/kg) in mice. Injections were administered on gestational days (GDs) 1 to 5, the period including implantation which is sensitive to hormonal balance. The effects of ZEN or E2 were evaluated by comparing the number of live fetuses, their weight, and absorbed conceptuses on GD 18, with those in vehicle-treated controls. In addition, implantation, embryos in the oviducts and those in uteri without implantation sites were investigated on GD 5. In mice treated with the highest dose of ZEN or E2, decidual responses and plasma progesterone concentrations were measured on GDs 5 and 6, respectively, and delayed implantation was investigated on GDs 9 and 14. The results showed that treatment with ZEN, in a manner similar to that seen for E2, led to obstruction of essential processes for establishing and maintaining pregnancy, such as embryo migration from oviducts to uteri, the decidual response, and activation of luteal function. Zearalenone also induced delayed implantation and loss of conceptuses and at low doses caused a retarded growth of the fetuses after normal implantation. It was therefore concluded that ZEN causes multiple estrogenic toxic actions when administered during early pregnancy in mice.

Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis.

Gene expression of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGF-R) and the localization of the corresponding proteins in the canine testis were studied. Levels of mRNA expressions were determined by semiquantitative reverse transcription polymerase chain reaction in the testes of the peripubertal (4-6 months), young adult (3-4 years), advanced adult (7-8 years) and senescent (11-16 years) groups. The EGF-R mRNA level in the testes of the peripubertal group was significantly higher than those in the other groups, whereas there was no difference in EGF and TGF-α mRNA levels among groups. Immunohistochemical stainings for EGF, TGF-α and EGF-R in the testis revealed that immunoreactivity in the seminiferous epithelium and Sertoli cell was weak and nonspecific for the stage of spermatogenesis, and distinct staining was found in Leydig cells. These results suggest that the EGF family of growth factors may be involved in testicular maturation and function in the dog.

Positive correlation between patency and mRNA levels for cyclooxygenase-2 and prostaglandin E synthase in the uterine cervix of bitches with pyometra.

Factors involved in patency of uterine cervices in the bitch with pyometra remain to be clarified. This study examined relationship between patency and mRNA levels for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2 and prostaglandin E synthase (PGES) in the uterine cervix of bitches with pyometra. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Levels of mRNA expression were determined by semi-quantitative reverse transcription-polymerase chain reaction. The cervical patency was positively correlated with mRNA levels for COX-2 and PGES, but not those for iNOS and COX-1. The results suggest that gene expression of COX-2 and PGES may be involved in the regulation of patency in the uterine cervix of bitches with pyometra.

Clinical study report on milk production in the offspring of a somatic cell cloned Holstein cow.

This study examined two female offspring of a somatic cell cloned Holstein cow that had reproduction problems and milk production performance issues. The two offspring heifers, which showed healthy appearances and normal reproductive characteristics, calved on two separate occasions. The mean milk yields of the heifers in the first lactation period were 9,037 kg and 7,228 kg. The relative mean milk yields of these cows were 111.2% and 88.9%, respectively, when compared with that of the control group. No particular clinical abnormalities were revealed in milk yields and milk composition rate [e.g., fat, protein and solids-not-fat (SNF)], and reproductive characteristics of the offspring of the somatic cell cloned Holstein cow suggested that the cloned offspring had normal milk production.