Protection of CHO cells by transfer of survivin driven by ovarian-specific promoter OSP-2.

Chemotherapy is the major therapy for cancer in clinic. However, chemotherapeutic agents can harm the other tissues/organs besides cancer. Thus, there are great interests in protecting the innocents by the transfer of protective genes. There are two problems to be solved, one is the selection of protective genes and the other is the orientation of the exotic genes. Recent researches demonstrated that the principal mechanism of chemotherapeutics was through apoptosis. Hereby, introduction of anti-apoptosis genes might interrupt the processes of apoptosis to avoid side effect from chemotherapeutics. On the other hand, tissue-specific promoters, which control gene expression in a tissue-specific manner, might be an alternative tool to guarantee the location of target genes. In this research, we applied gene therapy to chemoprotection using anti-apoptosis gene survivin and ovarian-specific promoter OSP-2. The results showed that OSP-2 could specifically drive the expression of survivin in ovarian cells and survivin could protect cells via inhibiting apoptosis. This might put a light on the future of chemoprotective gene therapy.

[Effects of gonadotroph-releasing hormone analogues on follicle apoptosis in rats with chemotherapy-induced ovarian damage].

OBJECTIVE: To study the effects of gonadotroph-releasing hormone (GnRH) agonist (GnRH-a) and GnRH antagonist (GnRH-ant) on cyclophosphamide (CTX)-induced follicle apoptosis in female rats. METHODS: Thirty-six female Sprague- Dawley rats were randomized into 6 groups, namely normal saline (NS), CTX, GnRH-a+NS, GnRH-a+CTX, GnRH-ant+NS, and GnRH-ant+CTX groups. The rats were sacrificed between the first and second week after the treatments., and the follicle apoptosis was investigated using TUNEL assay and transmission electron microscopy. RESULTS: The apoptosis rate of the granulose cells in the follicles in late development was significantly higher than that in early follicles, and the apoptosis rate of the oocytes and granulose cells in rats with CTX treatment was significantly higher than that in rats without CTX treatment (P<0.05). The apoptosis rate of the granulose cells in GnRH-a groups (ranging from 33.40 - or + 4.59 to 73.25 - or + 5.35) was significantly higher than that in GnRH-ant groups (27.46 - or + 4.52 to 49.38 - or + 5.02, P<0.05), but there was no significant difference in the oocytes of early follicles between GnRH-a groups (23.48 - or + 4.25 to 36.15 - or + 4.23) and GnRH-ant groups (21.47 - or + 3.81 to 34.04 - or + 5.54, P>0.05). Electron microscopy revealed characteristic apoptotic changes of the oocytes in early follicles and granulose cells in early and late follicles. The apoptotic changes were especially typical in the granulose cells showing the formation of the apoptotic bodies, and the oocytes only showed chromatin condensation and aggregation. CONCLUSION: In the rat mode, GnRH-a promotes while GnRH-ant suppressed follicle apoptosis induced by CTX. GnRH analogues regulates mainly granulose cell apoptosis, but have little effect on oocyte apoptosis.

[Effect of anti-Müllerian hormone on P450 aromatase mRNA expression in cultured human luteinized granulose cells].

OBJECTIVE: To investigate the effect of anti-Müllerian hormone (AMH) on hormone secretion and P450 aromatase mRNA expression from cultured human luteinized granulosa cells. METHODS: Human luteinized granulose cells were derived from 10 patients treated by in vitro fertilization-embryo transplantation (IVF-ET) in the Second Affiliated Hospital of Sun Yat-sen University from June to December 2006. Granulose cells were divided into group A, B, C, D, E depending on different concentration of AMH, testosterone group and blank group. 1x10(-7) mol/L testosterone and 1, 5, 10, 20, 50 microg/L AMH were added into the culture medium of group A, B, C, D and E. 1x10(-7) mol/L testosterone was added into the culture medium of testosterone group while no other ingredient was added into the medium of blank group. Estrogen levels in supernates were measured at 24, 48, 72 hours after cell incubation. RT-PCR was performed to detect the P450 aromatase mRNA expression in group B, C, D, E and testosterone group at 72 hours after cell incubation. RESULTS: (1) Estrogen levels in supernates of granulose cell culture at 24, 48, 72 hours were (8.529+/-0.381)x10(4), (10.977+/-0.436)x10(4), (13.309+/-0.506)x10(4) pmol/L in group A, (7.027+/-0.276)x10(4), (9.167+/-0.300)x10(4), (10.794+/-0.555)x10(4) pmol/L in group B, (6.039+/-0.226)x10(4), (7.585+/-0.548)x10(4), (8.797+/-0.518)x10(4) pmol/L in group C, (5.118+/-0.460)x10(4), (5.716+/-0.496)x10(4), (6.205+/-0.667)x10(4) pmol/L in group D, (4.932+/-0.148)x10(4), (5.323+/-0.184)x10(4), (5.629+/-0.212)x10(4) pmol/L in group E. When compared with blank group [(0.001+/-0.001)x10(4), (0.006+/-0.003)x10(4), (0.029+/-0.011)x10(4) pmol/L], the statistical differences were observed in group A, B, C, D, E (P<0.01); when compared with testosterone group [(8.418+/-0.569)x10(4), (10.841+/-0.689)x10(4), (13.301+/-0.637)x10(4) pmol/L], the statistical differences were observed in group B, C, D and E (P<0.01); statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C (P<0.01). No significant difference was observed between group D and E (P>0.05). In group A, B, C, D, E and testosterone group, the estrogen levels at 24 hours after cell culture were significantly lower than those at 48 and 72 hours (P<0.01); statistical difference was observed between estrogen levels at 48 and 72 hours (P<0.01). No significant difference was observed among 24, 48 and 72 hours in blank group (P>0.05). (2) Relative ratios of intensity of P450 aromatase/beta-actin at 72 hours of cell culture in group B, C, D and E were 0.6148+/-0.0046, 0.5156+/-0.0012, 0.4698+/-0.0027 and 0.4282+/-0.0017, respectively, which were statistically lower than that in testosterone group (0.8224+/-0.0021, P<0.01); statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C (P<0.01). No significant difference was observed between group D and E (P>0.05). CONCLUSION: It is suggested that AMH might affect estrogen synthesis by inhibiting P450 aromatose activity so that lead to hyperandrogenism microenvironment in local ovary.

Cloning and transcriptional activity of a novel ovarian-specific promoter from rat retrovirus-like elements.

The long terminal repeats (LTRs) are the control centers for retrovirus gene expression, which possess all of the requisite signals. It has been proved that the LTRs of Moloney murine leukemia virus (MoMLV) could constitutively activate genes in diverse cell types. Recently, a retrovirus-like element, OSP-1 (ovarian-specific promoter 1), was extracted from rat ovary according to the LTRs of MoMLV, whose name was derived from the fact of ovarian-specific transcription. It was reasonable to speculate that the tissue-specificity was acquired through mutations and that there should be abound other mutants, active or inactive. In the present study, we isolated several homologous sequences to OSP-1 and detected their function. Consequently, one of them could also drive target gene expression specifically to ovarian cell lines and was named OSP-2 which shared 98% similarity to OSP-1. On the other hand, we picked up other two closest sequences and proved them inactive, which was 97% and 95% similar to OSP-1, respectively. Sequence analysis revealed the different mutations around/within the binding sites of transcriptional factors that might play important roles in tissue-specificity. In summary, we extracted a novel ovarian-specific promoter as well as other nonfunctional mutants, which in part shed light on the study of ovarian-specific transcription. In addition, it also provided a new tool in cancer gene therapy and to create transgenic animals.

[Plasma metastin in adolescent polycystic ovary syndrome.].

OBJECTIVE: This study was designed to investigate the correlationship between plasma metastin and pathogenesis of adolescent women with polycystic ovary syndrome (PCOS). METHODS: From Jan. 2006 to Jun. 2006, 42 PCOS patients including 19 adolescent women and 23 adults with syndrome were treated in Second Affiliated Hospital of Sun Yat-Sen University. According to the range of age, those patients were divided into 19 cases in adolescent group ( 19 years). Meanwhile, 20 adolescent women were matched as controls. Blood samples were collected between day 1 and day 5 of a spontaneous bleeding episode in the PCOS groups and a menstrual cycle of the controls. The levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), free T (FT), dehydroepiandrosterone sulfate (DHEAS), sex hormone-binding globulin (SHBG), insulin, glucose, and metastin were detected from day 1 to day 5 of spontaneous bleeding or withdrawal bleeding by progesterone. On the next day, oral glucose tolerance test (75 g) and insulin release test were performed on those above patients and controls. The area under curve (AUC), the ratio of fasting blood glucose to insulin (GIR) and homeostasis model assessment insulin resistance index (HOMA-IR) were calculated. RESULTS: (1) The level of hormone: the level of LH was in (12 +/- 7) U/L in adult group and (12 +/- 8) U/L in adolescent PCOS group, which were significantly higher than (6 +/- 4) U/L in controls (P < 0.05). The level of FT was (2.3 +/- 1.2) pmol/L in adult group, which was significantly higher than (1.3 +/- 0.8) pmol/L in adolescent group and (1.1 +/- 0.5) pmol/L in control group (P < 0.05). It was observed that the level of (3.1 +/- 2.7)micromol/L in adolescent group was significantly lower than (6.3 +/- 2.7) micromol/L in control group (P < 0.05). Meanwhile, the level of FAI of 5.6 +/- 4.1 in adult group was significantly higher than 3.0 +/- 1.3 in control group (P < 0.05). No significant difference in FSH, T and SHBG levels among three groups were observed (P > 0.05). (2) Metastin and metabolism: Both the levels of fasting blood insulin, 2-hour insulin and AUC of insulin were (13 +/- 7) mU/L, (88 +/- 59) mU/L and (133 +/- 80) mUxL(-1)xmin(-1) in adolescent group, which were significantly higher than (7 +/- 3) mU/L, (57 +/- 29) mU/L and (82 +/- 34) mUxL(-1)xmin(-1) in control group. The fasting blood insulin of (13 +/- 7) mU/L in adolescent group was significantly higher than (9 +/- 5) mU/L in adult group. The level of fasting blood glucose and 2-hour glucose were (5.01 +/- 0.44) mmol/L and (6.48 +/- 1.16) mmol/L in adult group, which were significantly higher than (4.68 +/- 0.29) mmol/L and (5.44 +/- 0.83) mmol/L in control group and (4.67 +/- 0.30) mmol/L and (5.93 +/- 1.44) mmol/L in adolescent group. The glucose AUC of (9.99 +/- 1.85) mmolxL(-1)xmin(-1) in adult group was significantly higher than (8.42 +/- 1.53) mmolxL(-1)xmin(-1) in control group (P < 0.05). HOMA-IR of 2.6 +/- 2.0 in adolescent group was significantly higher than 1.4 +/- 0.7 in control group. GIR of 10 +/- 8 in adolescent group was significantly lower than 16 +/- 10 in control group (P < 0.05). The metastin level of (0.25 +/- 0.19) pmol/L in adolescent group and (0.29 +/- 0.29) pmol/L in adult group were all significantly higher than (0.18 +/- 0.23) pmol/L in control group (PPh glucose were observed (r = 0.256, 0.286 and 0.267. P = 0.044, 0.025 and 0.043). CONCLUSIONS: The expression of metastin in adolescent PCOS women was significantly higher that of normal adolescent women. The increased level of metastin might be associated with pathogenesis of adolescent women with PCOS.

[Cyclophosphamide-induced rat ovarian damage and expression of gonadotropin-releasing hormone receptor in the damaged ovaries].

OBJECTIVE: To investigate ovarian follicular damage induced by chemotherapeutic agents and gonadotropin- releasing hormone receptor (GnRHR) expression in the damaged ovaries in rats. METHODS: Two groups of adult SD rats were subjected to intraperitoneal injection of a single-dose cyclophosphamide and saline, respectively, and 8 weeks later, the ovaries were taken for observing the ovarian damages. The distribution of GnRHR was detected with immunohistochemistry, and RT-PCR was used to determine the expression of GnRHR mRNA in the rat ovaries. RESULTS: Massive primordial follicular loss occurred in the ovaries of rats exposed to cyclophosphamide with also evident stromal ovarian blood vessel damages and focal fibrosis. Both the protein and mRNA expressions of GnRHR were detected in normal rat ovaries, but in rats exposed to cyclophosphamide, the expressions were significantly lowered in the ovaries (P<0.05). CONCLUSION: Low-level GnRHR expressions in the ovaries of rats with cyclophosphamide exposure suggest microenvironment disturbances in the damaged rat ovaries in advanced stage of chemotherapy.

[Effects of gonadotropin releasing hormone analogues on chemotherapy-induced ovarian function damage in rats].

OBJECTIVE: To investigate the effects of gonadotropin releasing hormone agonist (GnRH-a) and antagonist (GnRH-ant) on cyclophosphamide (CTX)-induced ovarian damage in rats. METHODS: Seventy-two Sprague-Dawley female rats were divided randomly into six groups, which received normal saline (NS), CTX, GnRH-a + NS, GnRH-a + CTX, GnRH-ant + NS, and GnRH-ant + CTX respectively. Levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E(2)) were measured successively by the enzyme-linked immunosorbent assay method, and half of the rats were killed in the first week and between the fourth and the fifth week after stop of medication, respectively to compare the weight of the ovaries and the number of the primordial follicles and the growth follicles. RESULTS: (1) Throughout experiment, the serum levels of FSH, LH and E(2) of the control group fluctuated slightly, while those in the CTX group kept rising. During medication treatment, compared with the control group [(118 +/- 16) microg/L, (350 +/- 35) microg/L] and the CTX group [(113 +/- 15) microg/L, (289 +/- 42) microg/L], the concentrations of LH [(42 +/- 8) - (47 +/- 7) microg/L, (31 +/- 5) - (36 +/- 7) microg/L] and FSH [(124 +/- 45) - (136 +/- 32) microg/L, (178 +/- 54) - (198 +/- 27) microg/L] in the GnRH-a groups and the GnRH-ant groups were maintained at low levels significantly and the levels of LH in the GnRH-ant groups were significantly lower than that in the GnRH-a groups, but the levels of FSH in the GnRH-ant groups were significantly higher than that in the GnRH-a groups (P < 0.05); and extremely different from the GnRH-a groups [(0.98 +/- 0.18) - (1.46 +/- 0.22) ng/L], the level of E(2) of the GnRH-ant groups [(3.58 +/- 0.43) - (3.98 +/- 0.74) ng/L] did not significantly decrease (P < 0.01). After stop of therapy, the concentrations of LH, FSH and E(2) in the GnRH-a groups and the GnRH-ant + NS group rose gradually and were similar to the levels of the control group (P > 0.05), but the levels of FSH, LH and E(2) of the GnRH-ant + CTX group rose obviously and were similar to the levels of the CTX group, especially the FSH , and the levels of LH and FSH of the GnRH-ant + CTX group [(156 +/- 12) microg/L, (520 +/- 44) microg/L] and the CTX group [(178 +/- 18) microg/L, (546 +/- 36) microg/L] were significantly higher than that of the other four groups [(121 +/- 15) - (132 +/- 13) microg/L, (335 +/- 35) - (359 +/- 26) microg/L] at the 4(th) - 5(th) week after stop of treatment (P < 0.05). (2) At the 1(st) week after stopping therapy, the GnRH-a + NS group [(12 +/- 5) mg/100 g]and the GnRH-a + CTX group [(18 +/- 8) mg/100 g] had the lowest weight of ovaries which was significantly different from the other groups [(30 +/- 9) - (37 +/- 8) mg/100 g, P < 0.05]. At the 4(th) - 5(th) week after stopping therapy, the GnRH-ant + CTX group [(22 +/- 6) mg/100 g] and the CTX group [(20 +/- 4) mg/100 g] had the lowest weight of ovaries which were significantly different from the other groups [(29 +/- 5) - (31 +/- 9) mg/100 g, P < 0.05]. (3) At the 1(st) week after stopping therapy, there were the largest number of primordial follicles [(824 +/- 45), (689 +/- 39)] and the lowest number of growth follicles [(15 +/- 1), (21 +/- 3)] in the GnRH-a + NS group and the GnRH-a + CTX group, but there were the lowest number of primordial follicles [(255 +/- 24), (343 +/- 29)] and the largest number of growth follicles [(110 +/- 21), (87 +/- 17)] in the GnRH-ant + CTX group and the CTX group. At the 4(th) - 5(th) week after stopping therapy, the number of growth follicles in the GnRH-a + NS group (58 +/- 11) and the GnRH-a + CTX group (56 +/- 16) recovered to almost the level of the control group (57 +/- 9, P > 0.05), but the number of all kinds of follicles declined significantly in the GnRH-ant + CTX group [(195 +/- 15), (36 +/- 12)] and the CTX group [(212 +/- 11), (36 +/- 9)] compared to the other four groups [(302 +/- 15) - (690 +/- 43), (44 +/- 12) - (58 +/- 11), P < 0.05]. CONCLUSION: In rat model, GnRH-a prevents the ovarian function damage induced by CTX, but GnRH-ant does not show similar protective effect.

[Cyclophosphamide-induced ovarian damage and stem cell factor expression in rat ovaries].

OBJECTIVE: To investigate the possible pathways for ovarian injury after administration of cyclophosphamide in rats. METHODS: Adult SD rats received a single injection of saline vehicle or chemotherapeutic agent cyclophosphamide, and 8 weeks later, the ovaries were removed, fixed and serially sectioned for pathological examination and ovarian follicle counting. The expression of stem cell factor (SCF) protein was evaluated by immunohistochemistry and immunoreactive score, and SCF mRNA expression determined by RT-PCR in rat ovaries. RESULTS: Cyclophosphamide had a detrimental effect on ovarian stromal function and lead to primordial follicle loss. Immunoreactive SCF antigens were expressed on the oocytes in the primordial and primary follicles of rat ovaries, and also in the granulosa cells of the secondary follicles and early antral follicles. There was a higher granulosa SCF, lower oocyte SCF and higher SCF mRNA level in the ovaries of the rats exposed to cyclophosphamide as compared with those in control rat ovaries (P <0.05). CONCLUSION: Altered SCF expression in the ovaries of rats exposed to cyclophosphamide can be helpful for understanding the mechanisms for chemotherapeutic drug-induced ovarian damage.

[Genetic diagnosis of Huntington disease].

We report here a simple and effective method to assess the CAG repeat size of HD gene for gene diagnosis of Huntington disease. Genomic DNA sequences in polymorphic CAG repeat HD gene was amplified by PCR with TaKaRa LA Taq DNA polymerase and GC buffer. To distinguish normal alleles from HD alleles, DNA fragments of affected alleles were recovered as templates for secondary PCR. The secondary PCR products were cloned into T vector for sequencing to determine CAG repeat size. A total of 20 normal individuals and 3 members from a HD pedigree were included in this study. Results showed that the CAG repeat numbers in 20 normal individuals and 3 normal alleles from the HD pedigree varied in normal range, while in 3 HD alleles, the copy numbers of CAG repeat were 39, 40, 41 respectively. There was no overlap between the copy number of the normal and affected alleles. In conclusion, the TaKaRa LA Taq DNA polymerase with GC buffer can be used to effectively amplified CAG repeat of HD gene, which combined DNA sequencing can diagnose HD accurately. In addition, these finding suggested that the dynamic mutation in HD gene responsible for the genetic defect in Chinese HD patients.

[Molecular cloning and expression in cryptorchid testis of SRG2 from a mouse testis spermatocyte apoptosis-related gene].

It was observed that the spermatogenic cells apoptosis dramatically increased in infertile man. Cloning of novel spermatogenic cell-specific gene related to apoptosis is of momentous physiological and pathological significance to illustrate the apoptosis mechanism and the biology process of spermatogenic cells. A novel mouse gene full-length cDNA sequence-SRG2 was identified (GenBank accession number AF395083), which was significantly changed in cryptorchidism, from a mouse testis cDNA library using a cDNA fragment (GenBank accession number BE644542) as an electronic probe. SRG2 was 1,088 bp in length. The putative protein encoded by this gene was 295 amino acids with a theoretical molecular weight of 33,579 kDa and isoelectric point of 9.64. The sequence shared no significant homology with any known protein in databases except TSARG2, with which its homology was 78%. RT-PCR showed that SRG2 was expressed significantly in testis. Using molecular beacon probe to examine the mRNA expression level of SRG2 gene in cryptorchid testis of various stages, we found that the gene was up-regulated distinctly. Therefore, we conclude that this gene plays an important role in cryptorchid testis.

[46,XY female sex reversal patient with a novel point mutation in the coding sequence of the SRY gene].

OBJECTIVE: To investigate the molecular mechanism of a Chinese patient with 46, XY sex reversal. METHODS: DNA fragments of the SRY gene from the typical XY female sex reversal patient and her father were amplified by polymerase chain reaction (PCR). The amplified PCR fragments were cloned into the pUCm-T vector, and direct sequencing was carried out on an ABI 377-3 automated DNA sequencer to detect the mutation. PCR-restriction enzyme digestion was applied to detect the results of DNA sequencing. RESULTS: A novel mutation of the SRY gene was identified in the XY sex reversal patient of this study. A T is replaced by an A in codon 129 at position +387, resulting in the replacement of the polar amino acid tyrosine (TAT) by the stop code (TAA) in the HMG-box, whereas her father was proved to have the wild-type sequence. Because the mutation introduced an enzyme site of MaeIII, the PCR-restrict enzyme digestion showed that there were three bands (131 bp,231 bp and 247 bp) in the patient, whereas two bands (131 bp and 478 bp) in normal man. It verified the results of sequencing analysis. The results after searching the Human Gene Mutation Database showed that this mutation was not described before and should be a new mutation. CONCLUSION: The novel mutation in SRY gene has provided valuable information for the understanding of molecular mechanism of the patient with 46,XY female sex reversal.