The present study was conducted to determine the effect of two human chorionic gonadotropin (hCG) treatments on the fertility of ewes which were estrus-induced and artificially inseminated during the non-breeding season. Suffolk (SF: n=68) and South-Down (SD: n=20) ewes were pretreated with a controlled internal drug release dispenser (CIDR) for 12 days and 500 IU equine chorionic gonadotropin 1 day before CIDR removal. Each breed of ewes were divided into 3 groups: Group I (n=31), an intramuscular injection of 100 IU hCG was given on Days 3, 4 and 5 after artificial insemination (AI) (Day 0=the day of AI); Group II (n=29), an injection of 300 IU hCG was given on Day 4; and Group III (n=28, Control), an injection of 0.6% saline was given on Day 4. Plasma progesterone (P4) concentrations were measured by enzyme-immunoassay in all ewes in the three groups at the time of insemination (Day 0), and Days 4, 8, 12, 16, and 20 after AI. On Day 8, the mean P4 concentrations in ewes treated with hCG (Groups I and II) were significantly higher (P<0.01) than in the control ewes (Group III). In Group II, the mean P4 values on Days 14 and 18 were also higher (P<0.05) than those in the control ewes. Pregnancy (Days 20 and 58) and lambing rates were not significantly different among the three groups (37.9%, 55.2% and 54.2% for lambing rates). Prolificacy was also not significantly different among the three groups (1.36, 1.75 and 1.62). These results indicate that the present hCG treatment did not enhance fertility of the inseminated ewes, although the treatment stimulates corpus luteum and increased P4 concentrations.
The purpose of this investigation was to examine whether boar spermatozoa possess specific binding sites for relaxin and to examine what effects relaxin actually exerts on their motility characteristics. Boar spermatozoa were collected from cauda epididymides. For the relaxin binding study, washed cauda epididymal spermatozoa were smeared on glass slides, and specific sites that bind relaxin were identified by sequential application of a biotinylated relaxin probe, antibiotine immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. For the motility characteristics study, cauda epididymal spermatozoa were diluted 300-fold in modified KRB + 2% cauda epididymal fluid (approximately 1 × 10 7 cell/ml). After preincubation for 30 min at 37 C in a CO2 incubator, the sperm aliquots were placed in culture dishes covered with mineral oil and mixed with porcine relaxin. The concentrations of relaxin and spermatozoa in the aliquot were 100 ng/ml and 5 × 106 cells/ml, respectively. The aliquots were incubated for a certain defined period under the same conditions. After 30, 60, 90 and 120 min of incubation, sperm motility was recorded using a videotape recorder. As an evaluation of motility characteristics, the percent motility, grade of forward progression and velocity were examined. The results of the relaxin binding study demonstrated that relaxin bound with specificity to the midpiece and tail. In addition, bindings were visible at the acrosome cap and neck of the head. The results of the motility study which examined the effects of relaxin on the motility characteristics of boar sperm showed that relaxin did not cause a significant alteration in percent motility, but it improved forward progression. Relaxin treatment for 90 min produced a significant 1.4-fold enhancement in sperm velocity compared to that of control. These results suggest that relaxin activates sperm motility through a specific receptor.
The binding patterns of 20 lectins in the testes of the greater Japanese shrew mole (Urotrichus talpoides) were investigated by light microscopy. Eleven lectins (WGA, sWGA, LEL, STL, Jacalin, PSA, LCA, BPA, Con-A, WGA and PHA-E) exhibited a positive reaction in the cytoplasm and acrosomal region of spermatids and in the cytoplasm of Sertoli cells. LEL, BSL-I, DSA, sWGA, BSL-I and PHA-L were intensely positive in the cytoplasm of Leydig cells. The binding patterns in the acrosomal region of spermatids were divided into three types. For LEL, ECL and STL, the reaction was detected in the acrosomal regions from Golgi to cap phase spermatids and disappeared in acrosome and maturation phase spermatids. For SBA, VVL, ECL and DSL, the reaction was detected in the acrosomal region from Golgi to acrosome phase spermatids. No reaction was recognized in the acrosome of maturation phase spermatids. In DBA and Jacalin, the cytoplasm of maturation phase spermatids was positive although no reaction was detected in the acrosomal region of all spermatids. sWGA, BPA and LEL exhibited a strong reaction in the plasma membrane of zygotene spermatocytes at stages X and XI. BSL-II revealed a strong reaction in the cytoplasm of spermatogonia.
Corticotropin releasing-hormone (CRH) is recognized as modulating luteinizing hormone releasing hormone (LH-RH) secretion at the medial preoptic area (MPOA), but the physiological significance of the effects of endogenous CRH on LH-RH secretion at the median eminence (ME) is not clear. To clarify the effects of CRH at ME, we used two animal models (adrenalectomized and restraint stressed rats) for hypersecretion of CRH and examined the effects of iv administration of CRH antiserum on LH secretion. Adrenalectomy for 2 weeks clearly increased plasma concentrations of ACTH and decreased plasma concentrations of LH in adult male rats. The iv injection of CRH antiserum attenuated the increased levels of plasma ACTH and restored the suppressed levels of plasma LH in adrenalectomized rats. In response to restraint stress, plasma concentrations of ACTH and PRL were increased and plasma concentrations of LH were decreased in adult male rats. The iv injection of CRH antiserum blocked the suppression of LH secretion induced by stress, but it did not have any effects on PRL secretion. These results suggest that immunoneutralization of endogenous CRH at the ME increases LH secretion probably mediated by LH-RH release and ME is probably one of the action sites of CRH on LH-RH secretion in addition to MPOA.
In cattle, there is no direct evidence for subcellular localization of relaxin. This study investigated the ultrastructural features of the cytoplasmic electron-dense granules of bovine corpora lutea from the 4th month of gestation to near term, and the immunolocalization of relaxin in these granules. Corpora lutea collected from 28 Holstein cows were fixed with Bouin's solution for light microscopy or with 4% paraformaldehyde containing 0.3% glutaraldehyde followed by 1% osmium tetroxide for electron microscopy. Immunocytochemical detection of relaxin was done using polyclonal rabbit antiserum raised against purified porcine relaxin, and was performed by the peroxidase anti-peroxidase method or the protein A-gold technique for light and electron microscopy, respectively. Light microscopic immunocytochemistry showed immunostaining for relaxin in large luteal cells but not in small luteal cells. In the 4th month of pregnancy, weakly positive immunostaining of a few large cells was noted. Subsequently, the number of relaxin-positive cells and the intensity of immunostaining both increased gradually from the 7th month of pregnancy to near term. Ultrastructural examination revealed that the large luteal cells contained two types of cytoplasmic electron-dense granules, one being small granules of 100-300 nm in diameter and the other being large granules of 500-1800 nm. The small granules were abundant by the 7th month of pregnancy, decreased in number by the 9th month, and were absent near term. These granules were observed in the process of being released by exocytosis from the cell surface of the large luteal cells, especially in 4th to 6th months of pregnancy. In contrast, large granules were rare in the 4th month and then increased gradually to reach a maximum near term, and no evidence of their release from the luteal cells was obtained. After incubation with relaxin antiserum and protein A-gold, gold particles indicating relaxin immunoreactivity labelled the dense cores of most of the large granules but did not label small granules. These results indicate that the large luteal cells are the source of relaxin in the bovine corpus luteum during pregnancy and that electron-dense large granules are the subcellular site of relaxin storage within these cells.
Cell fusion is an important process in current animal biotechnology. This study was designed to establish optimal parameters for the electrofusion of hamster 2-cell blastomeres and to examine the dynamic changes in the cytoskeletal distribution using fluorescence staining. Various electric fields (70-2000 V/mm) and durations (50-1000 μsec) of electric pulse were applied. Electric fields higher than 300 V/mm or of duration longer than 200 μsec caused disruption of the blastomere. Blastomere fusion occurred at 70-170 V/mm of 100 μsec duration, with high yields (91%). However, electrofused embryos did not develop. Fluorescence observations showed that the control 2-cell embryos possessed a dense network of microtubules around the nucleus and abundant microfilaments at the cell-to-cell contact region. In the fused embryos, two domains of cytoskeleton, consisting of both microfilaments and microtubules, concentrated around each nucleus were observed. In the embryos which failed to fuse, however, a thin layer of microtubules and very thick bundle of microfilaments appeared at the cell-to-cell contact region. The results suggest that blastomere fusion is accompanied with cytoskeletal reorganization and that dynamic changes of the cytoskeleton occur in different modes between fused and non-fused embryos.
To develop new tests to predict bull subfertility, a mucus penetration test, in which penetration of cervical mucus by spermatozoa is quantitated, was applied using frozen-thawed semen and a test to examine the ability of spermatozoa to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187 (A23187 stimulation test) was attempted. Frozen-thawed spermatozoa from 4 Japanese black bulls (2 fertile and the other 2 subfertile) were analyzed by standard semen analysis such as sperm concentration, motility, viability and morphology, the mucus penetration test and the A23187 stimulation test, and the relationship of the test results to fertility status was investigated retrospectively. The results of the standard semen analysis and the mucus penetration test did not reveal any remarkable differences among the bulls, showing no relationship to fertility status. However, when spermatozoa were stimulated with 3 mM Ca2+ and 1 μM A23187, the bulls all showed a time-dependent induction of the acrosome reaction but the subfertile bulls showed a slower and lower induction than the fertile bulls. These results suggest that A23187 stimulation test may be useful when abnormal sperm characteristics cannot be found by standard semen analysis or the mucus penetration test.