Gap junctions (GJs) are intercellular channels. Molecules with a molecular weight of 1 kDa or less can pass back and forth between adjacent cells through GJs. Communication between oocytes and the somatic cells that surround them via GJs is known to play key roles to initiate oocyte maturation in many vertebrates. However, little is known of the detailed functions of ovarian GJs during oocyte maturation in fish. The oocyte maturation of fish is induced by a maturation-inducing steroid (MIS). The sensitivity of oocytes to the MIS is known as oocyte maturational competence (OMC) and is induced by luteinizing hormone (LH). However, LH receptors are found on the surface of granulosa cells rather than oocytes. We therefore proposed that the LH signals received by granulosa cells were passed to oocytes via GJs. This review describes current knowledge of the role of GJs between granulosa cells and oocytes during the LH-induced acquisition of OMC in fish.
Since the first somatic cell cloned calves were born in Japan in 1998, more than 500 cloned cattle have been produced by somatic cell nuclear transfer and many studies concerning cloned cattle and their offspring have been conducted in this country. However, most of the results have been published in Japanese; thus, the data produced in this country is not well utilized by researchers throughout the world. This article reviews the 65 reports produced by Japanese researchers (62 written in Japanese and 3 written in English), which employed 171 clones and 32 offspring, and categorizes them according to the following 7 categories: (1) genetic similarities and muzzle prints, (2) hematology and clinical chemistry findings, (3) pathology, (4) growth performance, (5) reproductive performance, (6) meat production performance and (7) milk production performance. No remarkable differences in health status or reproductive performance were found among conventionally bred cattle, somatic cell cloned cattle surviving to adulthood and offspring of somatic cell cloned cattle. Similarities in growth performance and meat quality were observed between nuclear donor cattle and their clones. The growth curves of the offspring resembled those of their full siblings.
Chronic hypobaric hypoxia (CHH) induces a decrease in sperm output and spermatogenesis in male rats. The mechanisms that underlie these changes in testicular function are unknown and could involve changes in the hypophysis-gonad axis. We have tested the hypothesis that changes take place in the endocrine status (FSH, follicle stimulating hormone; LH, luteinizing hormone; testosterone) of rats subjected to CHH. Male Wistar rats were maintained under normobaric or hypobaric conditions (428 torr, 4,600 m). On days 0, 5, 15 and 30 post-exposure, 12 rats were anesthetized, their body weights were measured and blood samples were collected. The testicles were fixed in 4% formaldehyde and processed for histological analysis. In this time course, the FSH levels rose by day 5 post-exposure. On subsequent days, the FSH levels decreased in rats subjected to CHH with a tendency to remain higher than the normoxic group. The LH plasma levels decreased in rats exposed to CHH. Consistent with the decrease in LH levels, the plasma testosterone level decreased significantly after 30 days of CHH exposure. Integrated analysis of hormonal changes in rats subjected to CHH and the body dehydration that occurs in HH allows us to conclude that the effects of CHH on spermatogenesis may be partially related to changes in the hypophysis-gonad hormonal axis.
The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 × 105 sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.
Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 μm in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 ± 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.
In this study, we investigated the effects of administration of coumestrol during pregnancy on calcium (Ca) metabolism in post-delivery maternal and neonatal mice. From 6.5 to 16.5 days post coitus (dpc), pregnant females were administered daily doses of coumestrol (200 μg/kg body weight/day). One day after parturition, blood samples and the kidneys, liver, jejunum and duodenum were obtained from each of maternal mouse, and blood samples and the kidneys and liver were obtained from neonatal mice. Coumestrol did not have any significant effect on the Ca and inorganic phosphorus concentrations in the sera of the maternal and neonatal mice. No notable effects of coumestrol were observed in relation to Vitamin D receptor expression in the maternal and neonatal mice by immunohistochemical analysis. Coumestrol did not affect the Vitamin D receptor and epithelial calcium channel1 and 2 mRNA levels in any of the organs investigated. Enzyme histochemical analysis showed that coumestrol decreased intestinal alkaline phosphatase activity in the maternal jejunum and duodenum. In the duodenum, coumestrol decreased expression of intestinal alkaline phosphatase, c-fos and vascular endothelial growth factor at the mRNA level. However, we did not observe any significant effects of coumestrol on the expression of these genes. In conclusion, coumestrol decreased intestinal alkaline phosphatase activity in the small intestines of maternal mice at the level used in the present study, and the mechanisms underlying this effect are different for the jejunum and duodenum.
The present study was carried out to examine the activation and parthenogenetic development of pig oocytes after exposure to ultrasound in sorbitol media supplemented with different concentrations of Ca2+. The activation rates (68.8-75.6%) of oocytes exposed to ultrasound in media containing 0.1-1.0 mM Ca2+ were significantly (P<0.05) higher than those (54.3-58.3%) of oocytes exposed to ultrasound in media containing 0-0.05 mM Ca2+. When oocytes were exposed to ultrasound in media containing 0.1-0.5 mM Ca2+, the blastocyst formation rates (20.5-21.3%) were significantly (P<0.05) higher than those (3.3-6.0%) of oocytes exposed to ultrasound in media containing 0, 0.05 or 1.0 mM Ca2+. The results of the present study showed that the concentration of Ca2+ in the medium used for exposure to ultrasound affects the activation and parthenogenetic development of pig oocytes and showed that the optimal Ca2+ concentration is 0.1-0.5 mM.
The aim of our study was to investigate the effects of suckling on reproductive performance and metabolic status of obese (mean body condition score of more than 4.0 on a scale of 1-5) maternal Japanese Black cows during early postpartum period. We used 7 postpartum Japanese Black cattle. Four cows were suckled ad libitum (suckled) until completion of their first artificial insemination (AI), while 3 cows were not suckled at all because they were separated from their calves immediately after parturition (non-suckled). Body weight and plasma concentrations of metabolites and hormones were measured from wk 1 to 9 postpartum. Ovarian activity was detected using plasma progesterone concentration, and all cows received their first AI after application of the Ovsynch protocol at approximately 4 months postpartum. Although body weights of non-suckled cows increased during experimental period (P<0.05), those of suckled cows remained unchanged. Plasma concentrations of glucose of non-suckled cows were higher at wk 2 postpartum (P<0.05) and their levels of non-esterified fatty acid tended to be lower at wk 1 and 2 postpartum compared with suckled cows (P<0.1); however, these differences between groups were not observed with progression of postpartum period. In addition, plasma insulin concentrations of non-suckled cows were higher than those of suckled cows during experimental period (P<0.05). During sampling period (wk 0 to 9 postpartum), onset of normal ovarian cycle was observed in all non-suckled and 2 of 4 suckled cows, and it was delayed in other 2 suckled cows compared with non-suckled cows; however, 3 suckled cows conceived at the first AI after application of the Ovsynch protocol; none of non-suckled cows conceived at this time. Overall, we suggest that suckling seems to reduce increase of body weight after parturition, although it does not improve obesity, and influences conception despite delay in resumption of normal ovarian cyclicity in obese Japanese Black cows.
To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin α, βA and βB subunts was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin α, βA and βB subunts were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin α, βA and βB subunts were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.
Obtaining sufficient transgenic cells via selective cultivation of genetically manipulated somatic cells is difficult due to the limited number of cell divisions. Additionally, if irreversible mutations in a cell's chromosomes occur during selective cultivation and the cell is used as the nuclear donor, somatic cell nuclear transfer (SCNT) embryos often exhibit abnormal development. On the other hand, a SCNT method in which fetal cells derived from SCNT embryos are used as the nuclear donor (recloning method) is an effective technique for obtaining large quantities of transgenic cells. In this study, we compared the in vivo development rate of SCNT embryos produced from porcine α1-3 galactosyltransferase gene knockout (GTKO) cells by a recloning method with that of SCNT embryos produced without recloning from porcine GTKO cells (direct method). In the direct method, 557 and 462 cloned embryos were produced using two types of activation methods, the two-step activation (TA) method and the delayed activation (DA) method, and then transferred into 6 and 4 recipients, respectively, but no piglets were born from these recipients. In the recloning method, 956 and 1038 cloned embryos were produced using the TA and DA methods, respectively, and then transferred to 8 and 7 recipients, respectively. Two piglets were born from one recipient in the TA group and 6 piglets were born from 3 recipients in the DA group. This report indicates that the recloning method improved the developmental capacity of SCNT embryos reconstructed with gene-targeted somatic cells.
The aim of this study was to evaluate the estrogenic activity of tuberous samples of wild, phytoestrogen-rich Pueraria mirifica collected from 28 out of 76 provinces of Thailand by MCF-7 proliferation assay. The plant extracts were administered to MCF-7, ERα positive human mammary adenocarcinoma cell cultures, for 3 days at dosages of 0.1, 1, 10, 100 and 1,000 μg/ml and were compared with 17β-estradiol at concentrations of 10-12-10-6 M. The mean P. mirifica population at 1 μg/ml exhibited significant proliferation. Two plant samples exhibited levels of proliferation in MCF-7 that were similar to 17β-estradiol. The mean P. mirifica populations at 100 and 1,000 μg/ml exhibited significant cytotoxicity in MCF-7. Analysis of the estrogenic activity of puerarin, representative of major isoflavonoids in P. mirifica tubers, revealed proliferation in MCF-7 only at the highest dose (10-6 M) that was 102-105 times less active than 17β-estradiol. Puerarin and 17β-estradiol at concentration of 10-12-10-6 M exhibited no cytotoxicity in MCF-7.
To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the well-established markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose- and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization.
The present study was planned to study the effects of addition of different concentrations of catalase enzyme (0, 250, 500 and 1,000 IU/ml) to cooled dromedary camel semen extended with tris-yolk-fructose extender on semen quality during storage at 5 C for up to 5 days. Conception rates of she-camels artificially inseminated with whole fresh or extended cooled dromedary camel semen with or without 500 IU/ml catalase enzyme were also estimated. The results showed that addition of catalase enzyme at concentrations of 250 or 500 IU/ml to extended cooled dromedary camel semen significantly increased (P<0.01) the percentage of sperm motility and significantly decreased (P<0.01) the percentages of dead spermatozoa, sperm abnormalities and acrosomal damage. The highest (P<0.01) percentage of sperm motility was recorded with extended cooled dromedary camel semen supplemented with catalase enzyme at a concentration of 500 IU/ml, and the lowest (P<0.01) value was recorded with catalase enzyme at a concentration of 1000 IU/ml. On the other hand, the lowest (P<0.01) percentages of dead spermatozoa, sperm abnormalities and acrosomal damage of spermatozoa were recorded with extended cooled dromedary camel semen supplemented with 500 IU/ml, and the highest (P<0.01) values were recorded with catalase enzyme at a concentration of 1,000 IU/ml. Advancement of the storage time at 5 C significantly decreased (P<0.01) the percentage of sperm motility and significantly increased (P<0.01) the percentages of dead spermatozoa, sperm abnormalities and acrosomal damage of spermatozoa. Moreover, the conception rates of she-camels artificially inseminated with whole fresh, extended cooled dromedary camel semen free-catalase enzyme and extended cooled dromedary camel semen supplemented with catalase enzyme at a concentration of 500 IU/ml were 46.15, 22.22 and 37.50%, respectively. In conclusion, the results show that addition of catalase enzyme at a concentration of 500 IU/ml to semen extender can be used as an agent for prolongation of dromedary camel sperm cell survival during storage at 5 C.
In studies of male reproductive toxicity, measuring daily sperm production is a quite important criterion. However, the accuracy of the values measured by the basic protocol is still controversial. In order to enhance the homogeneity of countable testicular sperm/spermatid heads, this report introduces a new enzymatic method with a subsequent detergent treatment. The testis of rat was firstly homogenized in phosphate-buffered saline. The homogenate (buffer mix) was then treated with collagenase and trypsin, and then sodium dodecyl sulfate (SDS) was added to produce detergent-resistant sperm/spermatid heads (detergent mix). After examination by hemocytometer, the coefficient of variation (CV) of the number of sperm/spermatid heads was compared with that obtained from the buffer mix. In addition, a MicroCell chamber was applied to the examination, and the CV was compared with other cases. In both examinations, homogeneity was improved by the detergent mix preparation. Counting with the hemocytometer showed an increased number of sperm/spermatid heads compared with that of the buffer mix (P<0.001), and the CV was decreased (P<0.05). In addition, when the MicroCell chamber was applied, the numbers increased about 3-hold compared with that of the buffer mix (P<0.001). The CV of the detergent mix was 23.7%, while that of the buffer mix was 38.9%. These results clearly demonstrate that the new preparation protocol generated in this study can provide more actual and accurate values when measuring daily sperm production.