Fever in rats during normal and dehydrated conditions

The effect of endogenous pyrogen (EP, from rabbit) and endotoxin (Salmonella typhosa) on rectal temperature (Tre) was investigated in normal and dehydrated rats of both sexes. Intraperitoneal injection of either EP or endotoxin did not affect body temperature. In addition, no changes in Tre were observed when endotoxin was injected intravenously in normally hydrated male rats, but significant falls in Tre occurred in normal female rats. However, intravenous injection of EP produced fever in both sexes, but females generally showed smaller responses. A second intravenous injection of endotoxin, given 3 days after the first injection, always produced fever in normally hydrated rats. The pattern of this febrile response was monophasic. In contrast to the response in normal rats, intravenous endotoxin produced significant fevers with a biphasic pattern in dehydrated rats of either sex, but the febrile responses of male rats were greater than those of female rats. On the other hand, there were no significant differences between febrile responses to intravenous EP exhibited by normal and dehydrated animals. These results show that rats of both sexes possess physiological mechanisms capable of producing a fever following intravenous injections of EP.     

Changes in the lipid-accumulating cells of the liver during experimental hypervitaminosis A

Changes of the fat-storing cells (FSC) in the liver have been studied after oil solution of vitamin A have been injected to intact rats, when Kupffer's cells have been blocked with colloid silver and stimulated with prodigiosan. During these experiments (on the 4th, 7th, 14th days) the amount of FSC does not change, and their size increases. The blockade of Kupffer's cells does not influence the dynamics of the FSC changes, and their stimulation contributes to lipid accumulation in their cytoplasm and to earlier revealing of these elements (on the 2d day of the experiment). In parallel with increasing size of FSC, fatty infiltration of hepatocytes disappears and reticular fibers accumulate in the perisinusoid space. Participation of FSC in lipid metabolism and in synthesis of the III type collagen is proved, as well as existence of intercellular interactions of these elements with endotheliocytes, Kupffer's cells and hepatocytes.     

Secretion profiles from in vitro cultured follicles, isolated from fresh prepubertal and adult mouse ovaries or frozen-thawed prepubertal mouse ovaries

In vitro folliculogenesis could be a new technology to produce mature oocytes from immature follicles that have been isolated from cryopreserved or fresh ovarian tissue. This technique could also be a tool for evaluation of oocyte quality and/or for determination of follicular parameters during follicular growth. Our objective was to characterize in mice the secretion profiles of follicles that had been isolated mechanically during in vitro follicular growth and in relation to the growth curve. Early preantral follicles from fresh prepubertal and adult mouse ovaries or frozen-thawed prepubertal mouse ovaries were cultured individually in microdrops under oil for 12 days. Each day, two perpendicular diameters of the follicles were measured. From day-3 to day-12 of culture, culture medium was collected and preserved for determination of inhibin B, anti-Müllerian hormone (AMH) and estradiol levels. At the end of the culture, after maturation, the status of the oocyte was evaluated. Follicular growth and their individual hormone production did not always correlate. Inhibin B was never secreted from follicles of less than 200 μm diameter, whether the follicles were examined when fresh or after freezing-thawing. Estradiol secretion was never observed in frozen-thawed follicles. AMH was mainly secreted between day-3 and day-9. Despite similar morphological aspects at the start of culture, follicles selected for in vitro folliculogenesis were found to be heterogeneous and differed in their ability to grow and to produce hormones, even if they had similar growth curves. Follicles from frozen-thawed ovaries developed slowly and produced fewer hormones than freshly collected follicles.     

Successful cryopreservation of mouse ovaries by vitrification

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.     

Craniofacial dysmorphogenesis following hypervitaminosis A in mice

Malformations of the temporomandibular joint (TMJ), zygomatic arch, middle ear ossicles, and mandibular musculature following hypervitaminosis A were described in fetal mice. Pregnant mice (Mus musculus) were each given a 0.2-ml solution of corn oil containing 10,000 IU of retinol palmitate by gavage on day 8.7. Thirty-eight fetal heads were collected and hemisected. The left hemiheads were serially sectioned, stained, and examined histologically. Right halves were cleared and double-stained with alizarin red and alcian blue. The craniofacial morphologies of the test mice were found to vary from normal to maximally involved (feature unidentifiable) even among littermates. Both inter- and intralitter variation were discussed. Errors in chondrogenesis were determined to have produced the variety of dysmorphologies observed. Changes in Meckel's cartilage affected both the TMJ and ossicles; the presence of ectopic cartilages affected the zygomatic arch; and the musculature was affected secondary to skeletal system changes. Several modes of vitamin A interference leading to craniofacial dysmorphogenesis have been proposed in the literature. It was determined that proposals implicating altered cellular differentiation were the most compatible with the in vivo data recorded here and elsewhere.     

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.     

大鼠卵巢不同功能阶段时c-fos表达的动态变化

目的和方法：本文用免疫组织化学方法检测了成年大鼠卵巢于不同的功能阶段和未成年大鼠外源性激素诱导的卵巢于不同的功能阶段c-fos表达的变化以及与血清E2和P含量的相关关系。结果：在成年大鼠动情前期、动情期卵巢的间质腺和基质中及动情间期和妊娠期卵巢的黄体细胞和基质中有c-fos表达,c-fos蛋白阳性信号的表达范围和表达强度在动情前期卵巢较大,动情间期和动情期卵巢较低,妊娠期卵巢最大,这种变化与血清E2和P含量的变化呈明显的正相关关系。未成年大鼠,用DES使卵泡发育至窦前期时,在卵巢中未检测到有c-fos蛋白的表达,用PMSG使贸泡发育至排卵前期时在卵巢的间质腺和基质中有c-fos表达,用PMSG和hCG后4天形成的早期黄体化卵巢c-fos在黄体细胞和基质中的表达明显升高,而于用hCG后9天形成的晚期黄体化卵巢c-fos表达明显下降,这种变化也与血清E2和P含量的变化呈明显的正相关关系。结论：c-fos与卵泡发育、排卵、黄体形成和退化等功能密切相关。     

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.