We investigated DNA synthesis during the 1-cell stage of reconstituted mouse embryos. Nuclei from 2-cell embryos were transferred at either the G1/S or G2 stage of the cell cycle. To define the S phase period in normal embryos, it was first examined that DNA synthesis in in vitro fertilized and parthenogenetic embryos during the first and second cell cycles. The results showed that DNA synthesis during the first cell cycle began between 6 and 8 h and finished between 12 and 14 h after insemination. In the second cell cycle the DNA synthesis occurred between 1 and 4 h after cleavage. We obtained G1/S phase nuclei using aphidicolin, DNA synthesis inhibitor. The DNA synthesis of the 2-cell embryos was significantly inhibited by exposure to aphidicolin and initiated soon after release from it. DNA synthesis during the first cell cycle of nuclear transferred embryos differed depending upon the cell cycle of the ooplast and the donor nucleus. When a G2 nucleus was fused with an non-activated ooplast and then it was activated, DNA synthesis proceeded as seen in parthenogenetic 1-cell embryos. While the DNA synthesis was not seen when G2 nucleus was transferred into an activated ooplast. An activated ooplast received a G1/S nucleus the DNA synthesis started soon after fusion and it continued for a longer period. These results showed that DNA was synthesized in the 1-cell embryos containing transferred nuclei in cell cycles of an ooplast and a donor nucleus dependent manner.
This study was carried out to examine, by the reverse transcription-polymerase chain reaction (RT-PCR), whether growth factor mRNAs [epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF) and platelet-derived growth factor A chain (PDGF-A)] express in porcine cumulus-oocytes complexes (COCs) during maturation in vivo and in vitro. Porcine oocytes or their surrounding cumulus cells were analyzed immediately after collection (0 h), at 12, 24, 36 and 42 h of in vitro maturation, and at 42 h of hCG injection in eCG primed gilts, respectively. EGF, IGF-I, bFGF and PDGF-A mRNAs were detectable in both oocytes and cumulus cells at all stages of in vitro maturation. TGF-α mRNA was detectable in cumulus cells at all stages of in vitro maturation, whereas TGF-α mRNA in oocytes was not detected throughout in vitro maturation. In addition, the expression patterns of those genes in COCs matured in vivo were entirely consistent with those in COCs matured in vitro. These results provide the evidence that several growth factor mRNAs are present in both oocytes and cumulus cells during maturation and support the concept that those factors are involved in the regulation of porcine oocyte maturation.
To examine the effect of mitochondrial DNA (mtDNA) on the developmental ability of mouse embryos, we investigated using the mice from standard and the mtDNA-congenic strain carrying different type of mtDNA under the same nuclear genetic background. When the 2-cell embryos (day 2 after hCG injection) derived from standard strain C57BL/6 (B6) carrying Mus musculus domesticus type mtDNA and from mtDNA-congenic strain C57BL/6.mtSPR (B6-mtSPR) carrying Mus spretus type mtDNA were used to compare the developmental ability in vitro, the percentage of embryos developed to blastocyst of B6-mtSPR strain (34.7%) showed significantly lower than that of B6 strain (93.8%) at day 6 after hCG injection. The results from reciprocal cross mating between B6 and B6-mtSPR to examine the effect of paternal mtDNA indicated that F1 embryos from B6-mtSPR females (38.7%) also exhibited lower developmental ability to blastocysts than those from B6 females (86.7%). When B6 and B6-mtSPR embryos were cultured in the low oxygen (5% O2) condition, development of B6-mtSPR embryos (96.7%) to the blastocyst stage was greatly improved to the level comparable to that of B6 embryos (97.4%). These results suggest that developmental ability of the embryos may be influenced by the type of maternally derived mtDNA through mitochondria respiratory pathway, but a possible involvement of incompativility among nuclear and mitochondrial genome and cytoplasmic factors on preimplantation development still remains to be resolved.
Progesterone has a facilitating effect on luteinizing hormone (LH) surges induced by estrogen in ovariectomized (OVX) rats. The aim of the present study was to elucidate the involvement of excitatory amino acids such as glutamate and aspartate, and noradrenaline (NA) in the mediobasal hypothalamus/median eminence (MBH) region in a progesterone-enhanced LH surge. Ovariectomized animals were implanted with a Silastic tubing containing crystalline estradiol (E2) for 2 days, and some of them received sc injection of progesterone (4 mg) at 0900 h on the day of sampling. The MBH was perfused using the push-pull perfusion method to determine the levels of glutamate, aspartate, glycine, glutamine, and NA during the LH surge. Blood samples were collected through an atrial cannula to determine plasma LH levels. In animals treated with both E2 and progesterone, levels of glutamate and glutamine significantly (P<0.01) increased simultaneously 1 to 2 h before the peak of LH surge, whereas those levels did not significantly change by E2 treatment alone. Levels of aspartate, glycine and NA showed no significant changes during steroid(s)-induced LH surges. These results suggest that the facilitating effect of progesterone on estrogen-induced LH surges might be mediated, at least in part, by glutamate released at the MBH where the majority of LHRH nerve terminals are distributed.
Ovariectomized goats were treated with subcutaneous implants containing ovarian steroid hormones and their effects on ingestive behavior and plasma metabolite concentrations were examined along with those on reproductive function. Sequential administrations of progesterone (P) and estradiol (E) reproduced steroidal milieu during the luteal through the follicular phases. Around 35 hours after the commencement of the E administration, estrous behavior appeared, and almost simultaneously the luteinizing hormone surge occurred. During the estrous period, amount of food intake and duration of rumination markedly decreased, whereas plasma concentrations of glucose and free fatty acid increased. These changes lasted until the E-implant was removed. The E administration without P pretreatment induced similar changes in reproductive, ingestive and metabolic parameters, although the occurrences of these were all advanced. In contrast, the administration of P alone had no such effect on any parameter. These results demonstrate that estrous behavior is accompanied by changes in feeding behavior and metabolism, and suggest that E is playing essential roles to prepare female for mating by coordinating diverse physiological functions in the goat.
To examine the hypothesis that whether nitric oxide (NO) in the brain is involved in actions of estrogen on reproduction and ingestion in ruminant species, three experiments were conducted using ovariectomized (ovx) or ovx plus estradiol (E) treated goats. In experiment 1, distributions of cells containing estrogen receptor (ER) or NO synthase (NOS) in the hypothalamus were determined immunohistochemically, which revealed that both ER and NOS positive cells were localized abundantly in the ventromedial nucleus of the hypothalamus (VMH). In experiment 2, several doses of sodium nitroprusside (SNP), an NO donor, was administered into the unilateral VMH of ovx goats immediately before the presentation of food. Injection of 1.5 μg SNP resulted in an abrupt decrease in food intake during the first 60-min of a 240-min feeding period. However, higher doses of SNP had no effect on food intake. In experiment 3, on the other hand, NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, was injected into the bilateral VMH of animals which had been treated with E. Although following the vehicle injection food intake was significantly suppressed by E as was seen in the VMH intact animal, this E-induced decrease in food intake was prevented by administering L-NMMA. This NOS inhibitor, on the other hand, altered neither the timing of the luteinizing hormone surge nor estrous behavior. These data suggest that NO in the VMH is involved in mediation of suppressive effect of E on ingestive behavior in the goat.unctions in the goat.
The present study examined the effect of cysteine and cystine associated with cumulus cells in maturation medium on oocyte glutathione (GSH) concentration at the end of maturation culture (for 48 h) and male pronuclear (MPN) formation following in vitro fertilization of pig oocytes. When cumulus-enclosed oocytes were cultured in a serum-free medium containing 0.57 mM cysteine or cystine, their GSH concentration and incidence of MPN formation were higher than when they were cultured without cysteine and cystine. Supplementation with cysteine during the final 24 h of culture resulted in an increased GSH concentration of oocytes and a higher incidence of MPN formation, regardless of the presence of cumulus cells, than in those cultured without cysteine or cystine. The same high incidence of MPN formation was observed even when oocytes were denuded after 36 h of culture and exposed to cysteine for only 12 h. In contrast, when oocytes were denuded after 24 h of culture and exposed to cystine, neither GSH synthesis nor MPN formation was improved, but both parameters did improve when oocytes were not denuded. Exposure of cumulus-enclosed oocytes to cystine from 36 h of culture did not promote MPN formation. These results indicate that cumulus-free pig oocytes can synthesize GSH in the presence of cysteine and that the presence of cumulus cells is essential for maintaining a high concentration of GSH in oocytes in the presence of cystine.
Two trials were conducted to investigate a simple superovulation method for Suffolk ewes during the breeding season. The method involves a single injection of porcine follicle-stimulating hormone (pFSH) combined with equine chorionic gonadotropin (eCG) and was evaluated by measuring the concentrations of plasma hormones (estradiol-17β(E2), progesterone (P4), luteinizing hormone (LH) and follicle-stimulating hormone (FSH)). The ewes were treated with a vaginal sponge containing synthetic steroid with progestative action (P sponge) for 12 days in both trials. In Trial 1 (conducted in 1995), 18 ewes received a single injection (F1) or multiple injections (F6) of a total of 20 mg pFSH, and in combination with an injection of 500 IU eCG (F1+P, F6+P). The injections were conducted on Day -2 (Day 0 was the day of P sponge removal). In trial 2 (conducted in 1996), 24 ewes were given single injections of pFSH and eCG at different times; (i) both hormones on Day -2 (F-2/P-2), (ii) pFSH on Day -2 and eCG on Day -1 (F-2/P-1), and (iii) both hormones on Day -1 (F-1/P-1). One hundred micrograms of an analogue of gonadotropin-releasing hormone (GnRH) was administered on Day 1 in Trial 1 and at the detection of estrus in Trial 2. Frozen-thawed semen was inseminated in utero using a laparoscope at 30 h and 38 h after P sponge removal in Trial 1 and at 18 hours after the detection of estrus in Trial 2. Blood samples were collected from the jugular vein. E2, P4, LH and FSH were assayed by second-antibody enzyme immunoassays. F1+P showed similar endocrine profiles to those of F6 and F6+P with a preovulatory E2 peak after P sponge removal, but F1 did not show a clear preovulatory E2 peak. The E2 concentration in F-2/P-1 rose biphasically; there was a rise just after injection of pFSH and then another rise after P sponge removal. Simultaneous injections of 20 mg pFSH and 500 IU eCG may reduce the E2 elevation. We concluded that a good ovarian response comparable to the multiple injection method can be achieved by the present simple superovulation method.
Two trials were conducted to investigate the effects of a simple superovulation method using a single injection of porcine follicle-stimulating hormone (pFSH) combined with equine chorionic gonadotropin (eCG) on ovarian responses and embryo qualities in Suffolk ewes during the breeding season. The ewes were treated with a vaginal sponge containing synthetic progestative steroid (P sponge) for 12 days in both trials. In Trial 1 (conducted in 1995), 18 ewes received a single injection (F1) or multiple injections (F6) of a total of 20 mg pFSH, and in combination with an injection of 500 IU eCG (F1+P, F6+P). The injections were conducted on Day -2 (Day 0, was the day of P sponge removal). In trial 2 (conducted in 1996), 24 ewes were given single injections of pFSH and eCG at different times: (i) both hormones on Day -2 (F-2/P-2), (ii) pFSH on Day -2 and eCG on Day -1 (F-2/P-1), and (iii) both hormones on Day -1 (F-1/P-1). One hundred micrograms of an analogue of gonadotropin-releasing hormone (GnRH) was administered on Day 1 in Trial 1 and at the detection of estrus in Trial 2. Frozen-thawed semen was inseminated in utero using a laparoscope at 30 h and 38 h after P sponge removal in Trial 1 and at 18 h after the detection of estrus in Trial 2. Embryos were recovered on Day 7 or Day 8. In Trial 1, the mean number of ovulations and the rate of normal embryos were 6.8, 16.7% for F1, 15.0, 36.0% for F1+P, 15.5, 27.0% for F6 and 20.3, 44.2% for F6+P. The F1+P group showed the results comparable to those of F6 and F6+P. In Trial 2, the mean number of ovulations were 17.8 for F-2/P-1, 13.1 for F-2/P-2 and 12.4 for F-1/P-1. The rate of normal embryos in F-2/P-1 (63.0%) was significantly higher than in F-2/P-2 (41.1%, P<0.05) and tended to be higher than in F-1/P-1 (43.6%, P=0.050). The rate of freezable embryos in F-2/P-1 (56.2%) tended to be higher than in F-2/P-2 (39.3%, P=0.057) and in F-1/P-1 (38.5%, P=0.076). It is concluded that the simple superovulation method of a single injection of 20 mg pFSH combined with 500 IU eCG was as effective as multiple injctions of pFSH, and in combined with eCG on superovulation of Suffolk ewes during the breeding season. Administration of pFSH on Day -2 and eCG on Day -1 was most efficient to induce superovulation in Suffolk ewes.
The golden hamster is a laboratory species that has provided a good experimental model for mammalian fertilization. The present study was undertaken to investigate the microtubule organization pattern during fertilization in the golden hamster. Mature unfertilized oocytes and zygotes obtained in vivo or in vitro were stained immunocytochemically with anti-α-tubulin monoclonal antibody. In mature unfertilized oocytes, microtubules were distributed as cytoplasmic foci of small aster-like arrays and in the meiotic spindle. After penetration by spermatozoa, microtubules in the meiotic spindle proceeded through the telophase configurations, while no obvious changes could be observed in the cytoplasmic astral microtubules. Throughout these early fertilization processes, incorporated spermatozoa were not associated with any detectable microtubules. At the pronuclear stage, astral microtubules in the cytoplasm moved to the peripheries of the male and female pronuclei. At prometaphase of the first mitotic division, the aster split and formed the poles for the mitotic spindle. After the first mitotic division, these asters developed into the interphase array of microtubules in the daughter blastomeres. In the second series of experiments, some oocytes were fertilized with hamster round spermatids by intracytoplasmic injection. The behavior of chromosomes and microtubules during fertilization in these oocytes were quite similar to those in oocytes fertilized with mature spermatozoa. These findings indicate that the centrosome in the hamster zygotes is completely of maternal origin.
Production and reproduction records of the Holstein dairy herd at Edinburgh University's Langhill farm were analyzed to study the incidence, possible risk factors and to assess the reproductive performance of cows with cystic ovaries. Risk factors investigated were whether a cow was from the genetically selected or control herd, 305 day milk yield equivalent of the previous lactation to the cystic one, and age/parity. For analysis of the consequences of cystic ovarian disease, cystic cows were individually matched to non-cystic herd mates for selection or control herd, age/parity, and month of calving. The cumulative lactational incidence of cystic ovarian disease was 8.1%. Selected herd cows were more at risk of developing cystic ovarian disease than control herd cows (p<0.05) and odds ratio 3.55. There was no significant association between high milk production in the previous lactation and COD (p<0.05). However, a significant association between increasing age/parity and cystic ovarian disease was found (p<0.01). The 305 day milk yield equivalent in the previous and cystic lactation for the cystic cows was not significantly different from the paired herd mates (p>0.05) respectively. However, the cystic cows produced significantly higher 305 day milk yield equivalent in the cystic lactation than the previous non-cystic lactation (p<0.001). The calving to conception and calving intervals were significantly longer in the cystic cows than the non-cystic paired cows (p<0.05) respectively. In conclusion, cystic ovarian disease causes an increase in the calving to conception and calving intervals. COD is common in older cattle. To understand risk factors more research needs to be done, especially high milk production prior to cystic ovarian disease diagnosis in the same lactation.
The primary objective of this study was to determine how the luteinizing hormone (LH) response to gonadotropin releasing hormone (GnRH) treatment after parturition correlates with the intakes of total digestible nutrient (TDN) and crude protein (CP), and the plasma concentration of glucose and cortisol during the postpartum period in high producing dairy cows (HPDC). The second objective was to determine whether GnRH analogue (GnRH-A) treatment induces ovulation and subsequently ovarian cyclicity in the acyclic cows. The day of normal parturition was designated DAY 0. Twenty-four multiparous Holstein cows were assigned randomly to one of three postpartum groups and accordingly received 200 μg of GnRH-A intramuscularly at either day 10 (DAY 10 group; n=9 postpartum acyclic cows), day 30 (acyclic DAY 30 group; n=4 postpartum acyclic cows: cyclic DAY 30 group; n=6 postpartum cows with functional corpus luteum (CL)) or day 60 (DAY 60 group; n=5 postpartum cows with functional CL). For the analysis of LH concentration, blood samples were collected at 15- or 30-min intervals until 8 h after the treatment. Plasma samples collected before the GnRH-A injection were assayed for LH, cortisol (CORTISOL) and glucose (GLUCOSE). In order to analyze TDN and CP intake until 9 weeks after parturition, 6, 2 and 4 cows were randomly selected from the DAY 10, acyclic DAY 30 and cyclic DAY 30 groups respectively. The CORTISOL (2.3 ± 2.3 ng/ml), GLUCOSE (45.5 ± 7.3 mg/dl), and plasma peak LH concentration after GnRH-A injection (27.6 ± 11.1 ng/ml), as well as the area under the LH response curve (AUC; 3250 ± 1505) and TDN intake at the week of GnRH-A injection as a percentage of requirement (67.8 ± 8.2 %) were smaller than other groups in the DAY 10 group (P<0.05). These items in the weeks following parturition tended to increase as the postpartum period progressed. From the results of DAY 10, we found only two significant positive correlation coefficients by rank (P<0.05) between CORTISOL and peak LH (r=0.72) and between CORTISOL and AUC (r=0.65). From the comparison between acyclic DAY 30 and cyclic DAY 30 groups, no clear correlation could be found between nutrition and pituitary LH release response to GnRH-A. In conclusion, the LH release induced by GnRH-A injection, plasma cortisol level and values of nutritional items in the weeks following parturition tended to increase as the postpartum period progressed. However in the same week after parturition, the LH response to GnRH-A treatment is correlated only with plasma cortisol level, and not with nutrition. Additionally, all the treated cows had single or multiple ovulations, and started or continued estrous cycles of normal length lasting for 18 to 22 days.
Immature epidermis, dermis and subcutaneous tissues were histologically observed in the fetal skin of rats on day 17 of pregnancy. The tissues had become thicker in the fetal skin by day 19, and formed the basement membrane. The strata corneum and granulosum were found in the fetal skin on day 21. The distance between the cutaneous surface and the vascular system increased with fetal development. None of the fetal skin contained any subcutaneous adipose tissue or sebaceous glands as in the maternal ones. Absorption ratios of radioactivities in the rat fetuses were assayed at 20 min after injecting 14C-phenol and 14C-4-tert-butylphenol into the amniotic fluid. The absorption of both compounds decreased with fetal development. The absorption of 14C-phenol was greater than that of 14C-4-tert-butylphenol in the rat fetuses in three stages. These results suggest that the decrease in the absorption with fetal development depends on the increase in the distance between the cutaneous surface and the vascular system, and on the formation of the stratum corneum. The lower absorption of the 14C-4-tert-butylphenol having high hydrophobicity could be because the fetal skin was without subcutaneous adipose tissue and sebaceous glands.
Polyclonal antibodies against the bacterially expressed mouse retinoblastoma gene product (pRb) were prepared and characterized to enable sensitive recognition of rodent pRb. The cDNA fragment corresponding to amino acids 237-595 of the mouse pRb sequence was expressed at a high level in E. coli as a fusion protein with glutathione S-transferase. Polyclonal anti-mouse pRb antibodies, MKS-1, were prepared by immunizing rabbits with the truncated pRb obtained from the fusion protein. MKS-1 sensitively recognized the hyper- and the hypophosphorylated pRbs not only in the rodent cells but also in primate cells by Western blotting following immunoprecipitation. An increase in the cellular content of total and hypophosphorylated pRbs during myogenic differentiation of mouse C2 cells was also detected quantitatively by MKS-1. Thus, MKS-1 is useful for the quantitative detection and the analysis of pRb in cell lines established from rodent tissues.