The Lateral Parabrachial Nucleus Inputs to the Lateral Hypothalamus Trigger Nocifensive Behaviors.

The lateral parabrachial nucleus (LPBN) is known to play a key role in relaying noxious information from the spinal cord to the brain. Different LPBN efferent mediate different aspects of the nocifensive response. However, the function of the LPBN â†’ lateral hypothalamus (LH) circuit in response to noxious stimuli has remained unknown. Here, we show that LPBN â†’ LH circuit is activated by noxious stimuli. Interestingly, either activation or inhibition of this circuit induced analgesia. Optogenetic activation of LPBN afferents in the LH elicited spontaneous jumping and induced place aversion. Optogenetic inhibition inhibited jumping behavior to noxious heat. Ablation of LH glutamatergic neurons could abolish light-evoked analgesia and jumping behavior. Our study revealed a role for the LPBN â†’ LH pathway in nocifensive behaviors.

An efficient approach to synthesize sterile allopolyploids through the combined reproduction mode of ameiotic oogenesis and sperm-egg fusion in the polyploid Carassius complex.

The association between polyploidy and reproduction transition, which is an intriguing issue in evolutionary genetics, can also be exploited as an approach for genetic improvement in agriculture. Recently, we generated novel amphitriploids (NA3n) by integrating the genomes of the gynogenetic Carassius gibelio and sexual C. auratus, and found gynogenesis was recovered in most NA3n females (NA3n♀I). Here, we discovered a unique reproduction mode, termed ameio-fusiongenesis, which combines the abilities of both ameiotic oogenesis and sperm-egg fusion, in a few NA3n females (NA3n♀II). These females inherited ameiotic oogenesis to produce unreduced eggs from gynogenetic C. gibelio and sperm-egg fusion from sexual C. auratus. Subsequently, we utilized this unique reproduction mode to generate a group of synthetic alloheptaploids by crossing NA3n♀II with Megalobrama amblycephala. They contained all chromosomes of maternal NA3n♀II and a chromosomal set of paternal M. amblycephala. Intergenomic chromosome translocations between NA3n♀II and M. amblycephala were also observed in a few somatic cells. Primary oocytes of the alloheptaploid underwent severe apoptosis owing to incomplete double-strand break repair at prophase I. Although spermatocytes displayed similar chromosome behavior at prophase I, they underwent apoptosis due to chromosome separation failure at metaphase I. Therefore, the alloheptaploid females and males were all sterile. Finally, we established a sustainable clone for the large-scale production of NA3n♀II and developed an efficient approach to synthesize diverse allopolyploids containing genomes of different cyprinid species. These findings not only broaden our understanding of reproduction transition but also offer a practical strategy for polyploidy breeding and heterosis fixing.

Changes in Ploidy Drive Reproduction Transition and Genomic Diversity in a Polyploid Fish Complex.

Unisexual animals are commonly found in some polyploid species complexes, and most of these species have had a long evolutionary history. However, their method for avoiding genomic decay remains unclear. The polyploid Carassius complex naturally comprises the sexual amphidiploid C. auratus (crucian carp or goldfish) (AABB) and the gynogenetic amphitriploid C. gibelio (gibel carp) (AAABBB). Recently, we developed a fertile synthetic amphitetraploid (AAAABBBB) male from C. gibelio by incorporating a C. auratus genome. In this study, we generated novel amphitriploids (AAABBB) by backcrossing the amphitetraploid male with the amphidiploid C. auratus. Whole-genome resequencing revealed the genomic changes, including recombination and independent assortment between homologs of C. gibelio and C. auratus. The fertility, sex determination system, oocyte development, and fertilization behaviors of the novel amphitriploids were investigated. Approximately 80% of the novel amphitriploid females recovered the unisexual gynogenesis ability. Intriguingly, two types of primary oocyte (with and without homolog synapsis) were discovered, and their distinct development fates were observed. Type I oocytes entered apoptosis due to improper synaptonemal complex assembly and incomplete double-strand break repair, whereas subsequent type II oocytes bypassed meiosis through an alternative ameiotic pathway to develop into mature eggs. Moreover, gynogenesis was stabilized in their offspring, and a new array of diverse gynogenetic amphitriploid clones was produced. These revealed genomic changes and detailed cytological data provide comprehensive evidence that changes in ploidy drive unisexual and sexual reproduction transition, thereby resulting in genomic diversity and allowing C. gibelio avoid genomic decay.

Prognostic impact of tertiary lymphoid structures in breast cancer prognosis: a systematic review and meta-analysis.

BACKGROUND: Tertiary lymphoid structures (TLSs), organizationally resemble lymph nodes, are frequently present in breast cancer (BCa). It is usually, but not always, associated with a positive prognosis or immunotherapy response in cancer patients. This meta-analysis was performed to assess the prognostic and clinical impact of TLSs in BCa. METHODS: We conducted a systematic search in PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, and WanFang Database to obtain eligible research data up to May 30, 2021. This meta-analysis is focusing on the studies evaluated the prognostic value of TLSs and the associated clinicopathologic indicators, related gene expression and survival. STATA software 16.0 software was used to assess the prognostic significance and clinical impact of TLSs. RESULTS: Nine studies involved with 2281 cases were incorporated in this meta-analysis, in which four of them evaluated the prognostic value of TLSs. There are 6 studies assessed the relationship of TLSs and 4 studies investigated the clinicopathologic parameters as well as the key gene expression, respectively. The results showed the presence of TLSs were predicting a better OS (HR = 0.61, 95% CI: 0.51-0.73, p < 0.001) and DFS (HR = 0.40, 95% CI: 0.17-0.93, p < 0.001) of BCa patients. It also revealed that the presence of TLSs was significantly correlated with tumor differentiation (p < 0.001), pTNM stage (p < 0.001), lymph node metastasis (p < 0.001), and TILs density (p < 0.001) of BCa, and the expression of Her2 (p < 0.001), ER (p < 0.001), PR (p < 0.001) and Ki67 (p = 0.009) of the tumor cell. CONCLUSION: Our results indicated that high levels of TLSs could predict a favorable prognosis for BCa. Moreover, the TLSs were significantly correlated with the clinicopathological indicators and the critical gene expression of BCa, indicating its potential clinical impact on BCa patients.

Changing trends of clinicopathologic features and survival duration after surgery for gastric cancer in Northeast China.

BACKGROUND: Through analyzing the data from a single institution in Northeast China, this study revealed the possible clinicopathologic characteristics that influence the prognosis of patients with gastric cancer (GC). AIM: To evaluate the changing trends of clinicopathologic features and survival duration after surgery in patients with GC in Northeast China, which is a high-prevalence area of GC. METHODS: The study analyzed the difference in clinicopathologic features and survival duration after surgery of 5887 patients who were histologically diagnosed with GC at the Harbin Medical University Cancer Hospital. The study mainly analyzed the data in three periods, 2000 to 2004 (Phase 1), 2005 to 2009 (Phase 2), and 2010 to 2014 (Phase 3). RESULTS: Over time, the postoperative survival rate significantly increased from 2000 to 2014. In the past 15 years, compared with Phases 1 and 2, the tumor size was smaller in Phase 3 (P < 0.001), but the proportion of high-medium differentiated tumors increased (P < 0.001). The proportion of early GC gradually increased from 3.9% to 14.4% (P < 0.001). A surprising improvement was observed in the mean number of retrieved lymph nodes, ranging from 11.4 to 27.5 (P < 0.001). The overall 5-year survival rate increased from 24% in Phase 1 to 43.8% in Phase 3. Through multivariate analysis, it was found that age, tumor size, histologic type, tumor-node-metastasis stage, depth of invasion, lymph node metastasis, surgical approach, local infiltration, radical extent, number of retrieved lymph nodes, and age group were independent risk factors that influenced the prognosis of patients with GC. CONCLUSION: The clinical features of GC in Northeast China changed during the observation period. The increasing detection of early GC and more standardized surgical treatment effectively prolonged lifetimes.

Prognostic significance of Borrmann type combined with vessel invasion status in advanced gastric cancer.

BACKGROUND: Borrmann classification (types I-IV) for the detection of advanced gastric cancer has been accepted worldwide, and lymphatic and/or blood vessel invasion (LBVI) status is related to the poor prognosis after gastric cancer. AIM: To evaluate the significance of Borrmann type combined with LBVI status in predicting the prognosis of advanced gastric cancer. METHODS: We retrospectively studied the clinicopathological characteristics and long-term survival data of 2604 patients who were diagnosed with advanced gastric adenocarcinoma at Harbin Medical University Cancer Hospital from January 2009 to December 2013. Categorical variables were evaluated by the Pearson's χ 2 test, the Kaplan-Meier method was used to identify differences in cumulative survival rates, and the Cox proportional hazards model was used for multivariate prognostic analysis. RESULTS: A total of 2604 patients were included in this study. The presence of LVBI [LBVI (+)] and Borrmann type (P = 0.001), tumor location (P < 0.001), tumor size (P < 0.001), histological type (P < 0.001), tumor invasion depth (P < 0.001), number of metastatic lymph nodes (P < 0.001), and surgical method (P < 0.001) were significantly correlated with survival. When analyzing the combination of the Borrmann classification and LBVI status, we found that patients with Borrmann type III disease and LBVI (+) had a similar 5-year survival rate to those with Borrmann IV + LBVI (-) (16.4% vs 13.1%, P = 0.065) and those with Borrmann IV + LBVI (+) (16.4% vs 11.2%, P = 0.112). Subgroup analysis showed that the above results were true for any pT stage and any tumor location. Multivariate Cox regression analysis showed that Borrmann classification (P = 0.023), vascular infiltration (P < 0.001), tumor size (P = 0.012), pT stage (P < 0.001), pN stage (P < 0.001), and extent of radical surgery (P < 0.001) were independent prognostic factors for survival. CONCLUSION: Since patients with Borrmann III disease and LBVI (+) have the same poor prognosis as those with Borrmann IV disease, more attention should be paid to patients with Borrmann III disease and LBVI (+) during diagnosis and treatment, regardless of the pT stage and tumor location, to obtain better survival results.

Sleep deprivation of rats increases postsurgical expression and activity of L-type calcium channel in the dorsal root ganglion and slows recovery from postsurgical pain.

Perioperative sleep disturbance is a risk factor for persistent pain after surgery. Clinical studies have shown that patients with insufficient sleep before and after surgery experience more intense and long-lasting postoperative pain. We hypothesize that sleep deprivation alters L-type calcium channels in the dorsal root ganglia (DRG), thus delaying the recovery from post-surgical pain. To verify this hypothesis, and to identify new predictors and therapeutic targets for persistent postoperative pain, we first established a model of postsurgical pain with perioperative sleep deprivation (SD) by administering hind paw plantar incision to sleep deprivation rats. Then we conducted behavioral tests, including tests with von Frey filaments and a laser heat test, to verify sensory pain, measured the expression of L-type calcium channels using western blotting and immunofluorescence of dorsal root ganglia (an important neural target for peripheral nociception), and examined the activity of L-type calcium channels and neuron excitability using electrophysiological measurements. We validated the findings by performing intraperitoneal injections of calcium channel blockers and microinjections of dorsal root ganglion cells with adeno-associated virus. We found that short-term sleep deprivation before and after surgery increased expression and activity of L-type calcium channels in the lumbar dorsal root ganglia, and delayed recovery from postsurgical pain. Blocking these channels reduced impact of sleep deprivation. We conclude that the increased expression and activity of L-type calcium channels is associated with the sleep deprivation-mediated prolongation of postoperative pain. L-type calcium channels are thus a potential target for management of postoperative pain.

Rosiglitazone ameliorates tissue plasminogen activator-induced brain hemorrhage after stroke.

OBJECTIVE: Delayed thrombolytic therapy with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier (BBB) breakdown after ischemic stroke and lead to catastrophic hemorrhagic transformation (HT). Rosiglitazone(RSG), a widely used antidiabetic drug that activates peroxisome proliferator-activated receptor-γ (PPAR-γ), has been shown to protect against cerebral ischemia through promoting poststroke microglial polarization toward the beneficial anti-inflammatory phenotype. However, whether RSG can alleviate HT after delayed tPA treatment remains unknown. In this study, we sort to examine the role of RSG on tPA-induced HT after stroke. METHODS AND RESULTS: We used the murine suture middle cerebral artery occlusion (MCAO) models of stroke followed by delayed administration of tPA (10 mg/kg, 2 hours after suture occlusion) to investigate the therapeutic potential of RSG against tPA-induced HT. When RSG(6 mg/kg) was intraperitoneally administered 1 hour before MCAO in tPA-treated MCAO mice, HT in the ischemic territory was significantly attenuated 1 day after stroke. In the tPA-treated MCAO mice, we found RSG significantly mitigated BBB disruption and hemorrhage development compared to tPA-alone-treated stroke mice. Using flow cytometry and immunostaining, we confirmed that the expression of CD206 was significantly upregulated while the expression of iNOS was down-regulated in microglia of the RSG-treated mice. We further found that the expression of Arg-1 was also upregulated in those tPA and RSG-treated stroke mice and the protection against tPA-induced HT and BBB disruption in these mice were abolished in the presence of PPAR-γ antagonist GW9662 (4 mg/kg, 1 hour before dMCAO through intraperitoneal injection). CONCLUSIONS: RSG treatment protects against BBB damage and ameliorates HT in delayed tPA-treated stroke mice by activating PPAR-γ and favoring microglial polarization toward anti-inflammatory phenotype.

The peripheral immune response after stroke-A double edge sword for blood-brain barrier integrity.

The blood-brain barrier (BBB) is a highly regulated interface that separates the peripheral circulation and the brain. It plays a vital role in regulating the trafficking of solutes, fluid, and cells at the blood-brain interface and maintaining the homeostasis of brain microenvironment for normal neuronal activity. Growing evidence has led to the realization that ischemic stroke elicits profound immune responses in the circulation and the activation of multiple subsets of immune cells, which in turn affect both the early disruption and the later repair of the BBB after stroke. Distinct phenotypes or subsets of peripheral immune cells along with diverse intracellular mechanisms contribute to the dynamic changes of BBB integrity after stroke. This review focuses on the interaction between the peripheral immune cells and the BBB after ischemic stroke. Understanding their reciprocal interaction may generate new directions for stroke research and may also drive the innovation of easy accessible immune modulatory treatment strategies targeting BBB in the pursuit of better stroke recovery.

The evolving role of neuro-immune interaction in brain repair after cerebral ischemic stroke.

Stroke is the world's leading cause of disability with limited brain repair treatments which effectively improve long-term neurological deficits. The neuroinflammatory responses persist into the late repair phase of stroke and participate in all brain repair elements, including neurogenesis, angiogenesis, synaptogenesis, remyelination and axonal sprouting, shedding new light on post-stroke brain recovery. Resident brain glial cells, such as astrocytes not only contribute to neuroinflammation after stroke, but also secrete a wide range of trophic factors that can promote post-stroke brain repair. Alternatively, activated microglia, monocytes, and neutrophils in the innate immune system, traditionally considered as major damaging factors after stroke, have been suggested to be extensively involved in brain repair after stroke. The adaptive immune system may also have its bright side during the late regenerative phase, affecting the immune suppressive regulatory T cells and B cells. This review summarizes the recent findings in the evolving role of neuroinflammation in multiple post-stroke brain repair mechanisms and poses unanswered questions that may generate new directions for future research and give rise to novel therapeutic targets to improve stroke recovery.

Preliminary screening and analysis of metastasis-related lncRNA and co-expressed papillary thyroid carcinoma mRNA.

The objective of the present study was to investigate the long non-coding RNA (lncRNA) and mRNA expression profiles that are associated with the invasion and metastasis of papillary thyroid carcinoma (PTC). Transwell invasion assays were used to screen three highly invasive sub-strains of the human PTC IHH4 cell line: IHH4-M1, IHH4-M2 and IHH4-M3. In addition, tumor-bearing nude mice were used to identify the invasive and metastatic capacity of the three sub-strains. Agilent lncRNA microarray chips were used to screen 795 differentially expressed lncRNAs and 788 differentially expressed mRNAs. A total of 10 lncRNAs and 10 mRNAs were randomly selected for RT-qPCR validation to confirm that the results were consistent with the microarray chips, suggesting that the results of the microarray chip analysis were relatively accurate. Gene ontology enrichment-based cluster analysis revealed that the differentially expressed genes were mainly associated with steroid biosynthesis, bioadhesion, intercellular adhesion and other metastasis-associated biological processes. The results of the pathway cluster analysis identified that the differentially expressed genes were associated with tumor metastasis-associated signaling pathways, including the cholesterol metabolic signaling pathway, the sterol regulatory element-binding protein signaling pathway and the integrin signaling pathway, suggesting that lncRNA may regulate PTC metastasis through various signaling pathways. The present study screened and constructed PTC metastasis-associated lncRNA and mRNA expression profiles, and it provides a molecular basis for the future study of high-risk molecular markers of PTC.

Cutaneous clear cell/signet-ring cell squamous cell carcinoma arising in the right thigh of a patient with type 2 diabetes: combined morphologic, immunohistochemical, and etiologic analysis.

BACKGROUND: The clear cell/signet-ring cell variant of cutaneous squamous cell carcinoma (cSCC) is extremely rare. Its carcinogenesis has consistently been linked to ultraviolet radiation and HPV in the literature. However, there is little definite information about the contribution of diabetes mellitus (DM) to cSCC. CASE PRESENTATION: A 78-year-old Chinese woman with type 2 DM presented with a mushroom-like lump in her right thigh. Histological findings revealed that the lesion was mainly composed of clear cells and signet-ring cells. The septa of vacuoles in cytoplasm displayed positivity for periodic acid schiff (PAS) and cytokeratins such as AE1/AE3, CK5/6, CK14, and CK19. Malignant cells did not express CK7, CK8, CK18, CK20, p16, p53, or c-erbB-2, and the Ki-67 index was less than 5 %. We further explored the etiology of clear cell/signet-ring cell cSCC using human papillomavirus (HPV) type-specific PCR and genotyping and confirmed that the patient was not infected with HPV. Nucleus positivity for p63 indicated the involvement of the p53 family in the lesion. Meanwhile, the expression of fibroblast growth factor receptor-2 (FGFR2), a downstream effector of p63, was upregulated in tumor cells. CONCLUSIONS: This study provides the first report on the clear cell/signet-ring cell variant of cSCC found in the right thigh of a patient with type 2 DM. Metabolic imbalance in addition to conventional pathogens such as UV and HPV may contribute to the development of the lesion via p63/FGFR2 axis.

Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle.

In this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy, respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%, P<0.01), but similar to that of ICSI group (68.5% vs. 65.5%, P>0.05). The 2-cell rate in NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P<0.01). Further studies testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to low efficiency of cloning.

NuMA distribution and microtubule configuration in rabbit oocytes and cloned embryos.

The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. Alpha-tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2-4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.

Dynamic changes in NuMA and microtubules in monkey-rabbit nuclear transfer embryos.

Previous reports have indicated that failure in cloning monkey is attributed to the removal of nuclear mitotic apparatus (NuMA) during enucleation and subsequent abnormal organization of mitotic apparatus. This study investigated the transformation and assembly of tubulin and NuMA protein during the first cell cycle of cloned monkey embryos reconstructed by using enucleated rabbit oocytes as recipients. After the oocyte fused with a fibroblast, extensive microtubule organization was observed around the introduced nucleus in most reconstructed embryos, suggesting the introduction of a somatic cell centrosome. A high proportion of fibroblast nuclei transferred into non-activated oocytes underwent premature chromosome condensation (PCC), transient spindle organization and chromosomes separation, followed by the formation of two pronucleus-like structures. In contrast, fibroblast nuclei in pre-activated ooplasm rarely underwent PCC, but formed a swollen pronucleus-like structure. Normal spindles were observed in about one third of the cloned embryos reconstructed by both methods. After transferring monkey fibroblasts into NuMA-removed enucleated rabbit oocytes, NuMA was localized in pseudo-pronuclei and gradually moved to mitotic spindle poles at the first mitotic spindle poles. NuMA antibody microinjection resulted in spindle disorganization and chromosome misalignment, but did not significantly affect early cleavage. Our findings indicate that: 1. NuMA in donor monkey fibroblast may contribute to form a normal spindle in enucleated rabbit oocyte; 2. when non-activated cytoplasts and pre-activated cytoplasts are used as recipients, the donor nuclei undergo different morphological changes, but yield similar early embryo development; 3. although abnormal spindle organization and chromosome alignment may cause low efficiency of animal cloning, these abnormalities do not significantly affect early cleavage.

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro.

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.

Full-term development of mouse eggs transplanted with male pronuclei derived from round spermatids: the effect of synchronization between male and female pronucleus on embryonic development.

Pronucleus transplanted mice have been produced, but their donor male pronuclei were derived from mature sperm and were completely synchronous with female pronuclei because both male and female pronuclei came from the same fertilized oocyte. The present study firstly produced male pronuclei by introducing round spermatids into enucleated mouse oocytes, then transferred the male pronuclei into mouse oocytes at three activation stages and finally compared the effect of three kinds of oocytes on the development of reconstructed embryos. Our results indicate that, in enucleated oocytes, mouse round spermatid nuclei can transform to male pronuclei in a higher proportion, and the synchronization between male and female pronucleus does not significantly influence the early cleavage but the later and full-term development of reconstructed embryos.

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse.

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.

The effects of different donor cells and passages on development of reconstructed embryos.

In order to study the effects of different donor cells and passages on development of nuclear transfer embryos, we constructed embryos by electrofusing several kinds of donor cells into enucleated M II oocytes from Kun Ming (KM) mouse. These cells include 2-cell embryonic blastomeres, KMW embryonic stem (ES) cells, fetal fibroblast, ear fibroblast, tail tip fibroblast, sertoil cells and spermatogonia. Meanwhile, we compared the effects of passage numbers of fetal fibroblast cells on developmental competency after nuclear transfer. We found that 7.4% of reconstructed embryos from 2-cell embryonic blastomeres and 0.7% from ES cell could develop to blastocyst in vitro; embryos from fetal fibroblast could only develop to morula stage with the rate of 0.2%; embryos from spermatogonia could only develop to 8-cell stage and the rate was 0.3%; embryos respectively from ear fibroblast, sertoli cell and tail tip fibroblast could only develop to 4-cell stage. Although 2-cell development rate of embryos reconstructed from fetal fibroblast in first passage was significantly lower than those from the 2nd, the 3rd and the 4th passage, embryos from different passages could develop to 8-cell stage except the 3rd passage. The result indicated that it is more difficult for terminally differentiated cell nuclei to be reprogrammed in enucleated M II oocytes than for low differentiated cell nuclei. The reason of low development rate from ES cells maybe that most of ES cells was at S stage of the cell cycle, which out of coordination with M II oocytes. We could conclude that culture and passage of donor cells might be benefit to nucleus reprogramming.

Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes.

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.