Development and fertility of ovaries in the B6.YDOM sex-reversed female mouse

When the Y chromosome of Mus musculus domesticus (YDOM) was introduced onto the C57BL/6 (B6) mouse background, half of the XY progeny (B6.YDOM) developed bilateral ovaries and female internal and external genitalia. We examined the fertility of the B6.YDOM sex-reversed female mouse. The chromosomal sex of the individual mouse was identified by dot hybridization with mouse Y chromosome-specific DNA probes. The results indicated that all XY females lacked regular estrous cyclicity although most were able to mate and ovulate after treatment with gonadotropins. When they had been ovariectomized and grafted with ovaries from the XX female litter mate, they initiated estrous cyclicity. Reciprocally, the XX female that had received XY ovarian grafts did not resume estrous cyclicity. Development of the XY ovary was morphologically comparable to the XX ovary until 16 day of gestation (d.g.), when most germ cells had reached the zygotene or pachytene stage of meiotic prophase. However, by the day of delivery (19 or 20 d.g.), no oocyte remained in the medullary cords of the XY ovary. In the control XX ovary, the first generation of follicles developed in the medullary region, and 5 delta-3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity appeared first in the stromal cells around growing follicles by 10 days after birth. In contrast, in the XY ovary, follicles were not formed in the medullary region, and 3 beta-HSDH activity appeared in epithelial cells of the oocyte-free medullary cords. Primordial follicles in the cortex region continued development in both the XX and XY ovaries. These results suggest that the XY female is infertile due to a defect inside the XY ovary. The prenatal loss of oocytes in the medullary cords may be a key event leading to abnormal endocrine function, and thereby, the absence of estrous cyclicity.     

Natural or hormone-induced sexual and social behaviors in the female brown lemming (Lemmus trimucronatus)

The behaviors of intact or ovariectomized, estradiol benzoate-treated or estradiol benzoate followed by progesterone-treated female brown lemmings were compared. Intact, diestrous females engaged in more social interactions with a male than did ovariectomized females (Experiment 1). In the first 5 min of a 1-hr mating exposure (Experiment 2, Test A) intact females in natural estrus engaged in more social and sexual behaviors than did ovariectomized females in estrogen-induced estrus. However, during the last 5 min of the 1-hr exposure (Test B) ovariectomized females receiving estrogen alone continued to show high levels of sexual activity with a male partner, while intact estrous females or females receiving estrogen followed by progesterone showed an apparent drop in sexual receptivity and an increase in aggressivity. Aggressive behaviors, as indexed by threat-leap behaviors on the part of the female may increase in the presence of progesterone. Declines in sexual activity, occurring within 1 hr of progesterone injection, were apparently dependent on the interaction of progesterone and copulatory events which may affect both the male and female.     

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1 ^-/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.     

Genotypic influences on reproductive aging of inbred female mice: effects of H-2 and non-H-2 alleles

The H-2 (major histocompatibility) complex of mice influences a variety of physiologic parameters. This study describes the influences of H-2 polymorphisms and other genetic influences on age-related changes (5-20 mo) in estrous cycles and fecundity. We monitored estrous cycles of virgin or retired-breeder mice of congenic strains on the background of C57BL/10Sn (B10):B10.BR/Sg (B10.BR) and B10.RIII/Sn (B10.RIII). For another comparison, we examined the C57BL/6J (B6) strain, which has the same H-2 haplotype as the B10. Estrous cycles were categorized by length during 10 mo of observations. From 5 to 15 mo of age, B10 and B10.RIII mice displayed a preponderance of 5-day cycles, B10.BR mice displayed a preponderance of 4-day cycles, and B6 mice had diminishing numbers of 4-day cycles. Age-related acyclicity differed with strain, particularly among retired breeders; B6 mice had an earlier onset and more rapid increase of acyclicity with age than the B10 congenic mice. Litters/female, maternal age at last litter, and total pups/female differed with strain; B10.BR and B10.RIII were similar and both had greater values than B10 mice. In conclusion, reproductive senescence of female mice was influenced by genes at the H-2 locus and elsewhere.     

The circadian basis of ovarian diapause regulation in Drosophila melanogaster: is the period gene causally involved in photoperiodic time measurement?

Females of a wild-type strain of Drosophila melanogaster (Canton-S), and of several clock mutants (period), were able to discriminate between diapause-inducing short days and diapause-averting long days with a well-defined critical daylength. The critical daylengths of a short-period mutant (pers) and a long-period mutant (perL2) were almost identical, both to each other and to that of Canton-S. The critical daylength of an arrhythmic mutant (perol), however, was about 3 hr shorter than that of Canton-S, and that of per- was about 5 hr shorter. Exposure of Canton-S females to Nanda-Hamner experiments, consisting of a 10-hr photophase coupled to a dark phase varying between 4 and 74 hr, showed (1) that the photoperiodic clock in D. melanogaster measures nightlength rather than daylength, and (2) that photoperiodic time measurement is somehow based on (or affected by) constituent oscillators in the circadian system. Nanda-Hamner results for the period mutants all showed similar profiles regardless of genotype, or the presence or absence of per locus DNA. These results suggest that photoperiodic induction and locomotor activity do not share a common pacemaker in D. melanogaster, and that the per gene is not causally involved in nightlength measurement by the photoperiodic clock, although flies in which the per locus is missing (per-) or defective (perol) show an altered critical value.     

Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein.

The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides. The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da). The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B. thuringiensis. Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain. The nprA3 strain produced insecticidal crystal proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal crystal proteins than did those of its isogenic counterpart. The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific crystal protein of B. thuringiensis. These results indicate that crystal protein stability and yield may be improved by deletion of specific proteases from B. thuringiensis.     

A New Intact Cell System for Determination of the Rate of Vitamin B6 Biosynthesis

To establish an intact cell system to determine the rate of vitamin B6 (B6) biosynthesis, conditions suitable for the reaction were examined. Cells of Escherichia coli B WG2, a pdxH mutant, were used for the reaction. Pyridoxal-starved cells of a stationary phase culture showed a negligible change of cell mass and a constant increase of B6 in the reaction. The rate of B6 biosynthesis was reliably determined with a 2-hr reaction initiated with cells of 0.10 to 0.15mg per ml. The influence of endogenous metabolites was completely abolished by the use of such a low concentration of the starved cells.

When glucose was used as a sole carbon substrate in the reaction system, the maximum rate of B6 biosynthesis was determined to be 0.5 to 0.6 nmol per mg cells per hr. The maximum rate increased to 0.8 to 0.9 nmol per mg cells per hr on supplementation of glycolaldehyde to the reaction mixture. The result led to the conclusion that the rate-limiting step in the reaction sequence of B6 biosynthesis lies the process of incorporation of the 5–5′ carbon fragment of the B6 molecule. This was also confirmed by a growth experiment with the wild type strain.     

Isolation and Characterization of a Temperature-Sensitive Circadian Clock Mutant of Neurospora Crassa

A new circadian clock mutant has been isolated in Neurospora crassa. This new mutation, called period-6 (prd-6), has two features novel to known clock mutations. First, the mutation is temperature sensitive. At restrictive temperatures (above 21°) the mutation shortens circadian period length from a wild-type value of 21.5 hr to 18 hr. At permissive temperatures (below 21°) the mutant has a 20.5-hr period length close to that of the wild-type strain. Second, the prd-6 mutation is epistatic to the previously isolated clock mutation period-2 (prd-2). This epistasis is unusual in that the prd-2 prd-6 double mutant strain has an 18-hr period length at both the restrictive and permissive temperatures. That is, the temperature-sensitive aspect of the phenotype of the prd-6 strain is lost in the prd-2 prd-6 double mutant strain. This suggests that the gene products of the prd-2 and prd-6 loci may interact physically and that the presence of a normal prd-2(+) protein is required for low temperature to ``rescue' the prd-6 mutant phenotype. These results, combined with our recent finding that prd-2 and some alleles of the frq gene show genetic synergy, suggest that it may be possible to establish a more comprehensive model of the Neurospora circadian clock.     

Histofunctional state of mouse ovaries in normal conditions and during hypervitaminosis A

Morphometry was used to analyze different histofunctional structures of mouse ovaries (follicles of varying maturation grades, yellow and atretic bodies, interstitial tissue) in health and hypervitaminosis A (80 000 IU). It was noticed that in the ovaries of female mice in health, there occurred consistent cyclic processes marked by the changes in the number and size of the morphofunctional structures during different stages of the estrous cycle. In hypervitaminosis A, female mice had no estrous cycle. The ovaries showed the impairment of the growth of the follicles and enhancement of atresia. As a result, the follicles did not reach the final stages of development, which led to the cessation of ovulation and formation of yellow bodies.     

Effects of antigens and lymphokines on early activation of single hapten-specific B lymphocytes

An assay was developed to monitor early activation of single fluorescein-specific B cells obtained from the spleens of nonimmunized adult mice by prefractionation on hapten gelatin. Early activation was assessed as a significant increase in the diameter of individual B cells after 24 hr in vitro. Significant enlargement of the single B cells was induced within 24 hr by either T-independent antigens acting alone or a crude source of B cell growth and differentiation factors (EL-BGDF-pik) acting alone. In contrast, T-dependent antigens acting alone were ineffective. When selected T-independent antigens and EL-BGDF-pik acted together, a greater number of B cells were induced to enlarge. B cell stimulatory factor 1 (BSF 1) behaved in a similar manner as EL-BGDF-pik, inducing early B cell enlargement both in the absence and more so in the presence of antigen. Both EL-BGDF-pik and BSF 1 enhanced the survival of single hapten-specific B cells during the 24-hr period. Interleukin 1 was unable to cause B cell enlargement when acting alone, although it was able to augment B cell enlargement induced by antigen. Interleukin 2 did not induce cell enlargement in either the presence or absence of antigen. Activation was demonstrated among cells of all sizes, regardless of the stimulus, although a greater response was demonstrated amongst the larger cell population. The addition of 3T3 filler cells enhanced early B cell activation and cell survival during the 24-hr period. The 24-hr B cell enlargement assay as applied to isolated single cells provides an unequivocal approach to the analysis of early B cell activation, adding a further parameter for the dissection of the precise roles of antigen and the various factors in the B cell differentiation pathway.     

Expression of bradykinin B2 receptor in the mouse ovary

The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mare's serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B(2) receptor mRNA was constitutively expressed. Bradykinin B(2) receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B(2) receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B(2) receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B(2) receptor mRNA level. The present data indicate that the level of B(2) receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.     

成体雌性小熊猫卵巢组织学观察

2000 年至2009 年,12 只固定于10% 福尔马林中非生殖系统疾病死亡的小熊猫卵巢组织,按常规组织学技术制作组织切片,HE 染色,光学显微镜观察。结果:(1)不同发情时期卵巢均有原始卵泡、初级卵泡和次级卵泡分布。发情期的卵巢未观察到典型的成熟卵泡和卵母细胞; (2)原始卵泡数量较少,初级卵泡数量较多,多数初级卵泡和大多数的次级卵泡都处在闭锁状态;(3)卵泡腔出现之前,卵母细胞的直径和卵泡直径同时增长;卵泡腔出现之后,卵母细胞直径增长较慢,卵泡直径增长较快; (4)不同发情时期的小熊猫卵巢均存在大量的间质腺细胞;(5)妊娠小熊猫和发情间期无妊娠小熊猫的卵巢均有发育正常的黄体;(6)卵泡细胞发育呈低柱状至柱状时出现透明带。结论:(1) 卵泡闭锁主要发生在初级卵泡阶段,仅少数卵泡能发育至次级卵泡;(2)卵母细胞和卵泡生长呈双相生长的趋势; (3) 不同发情时期的小熊猫卵巢间质腺都发达; (4)发情排卵后,非妊娠黄体与妊娠黄体维持的时间相似,证实了小熊猫存在假孕现象。     

猪链球菌2型srtF基因敲除突变株的构建及其毒力

利用同源重组基因敲除方法构建猪链球菌2型强毒株05ZYH33 srtF同源突变体。组合PCR、交叉酶切、RT-PCR结果均显示srtF突变体构建成功。突变株与强毒株的菌落形态、生长速率以及对小鼠的致病力均无显著性差异。小鼠竞争实验结果提示,突变株在心脏的定殖及感染能力显著减弱。成功构建的srtF突变株为进一步研究srtF的生物学功能奠定了基础。     

The ameliorative effects of Eurycoma longifolia Jack on testosterone-induced reproductive disorders in female rats

The objective of this research was to study the ameliorative effects of a standardized quassinoid-rich extract (TAF 273) of Eurycoma longifolia root on some reproductive disorders in female rats. An irregular estrous cycle and ovarian cystic follicles were induced in 21-day-old females by the daily administration of testosterone (10 mg/kg, sc) for three weeks. The hormone-treated rats exhibited persistent diestrous as well as ovaries containing cystic follicles. Upon treatment with TAF 273, fewer animals showed irregular estrous cycles and there was less follicular morphological damage. The reversal effect may be derived from the anti-estrogenic properties of the plant quassinoids.     

The Brucella abortus xthA-1 gene product participates in base excision repair and resistance to oxidative killing but is not required for wild-type virulence in the mouse model

Exonuclease III, encoded by the xthA gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with Escherichia coli xthA mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic xthA-1 mutant (designated CAM220) derived from virulent Brucella abortus 2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic xthA-1 mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The B. abortus xthA-1 mutant also displayed reduced resistance to killing by H2O2 and the ONOO(-)-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the xthA-1 gene product participates in protecting B. abortus 2308 from oxidative damage. Introducing a plasmid-borne copy of the parental xthA-1 gene into CAM220 restored wild-type resistance of this mutant to MMS, H2O2, and SIN-1. Although the B. abortus xthA-1 mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in C57BL/6 [corrected] mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the xthA-1 gene product participates in base excision repair and resistance to oxidative killing in B. abortus 2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.     

Daily changes in concentrations of prolactin in serum of prepubertal bulls exposed to short- or long-day photoperiods

Early temporal changes in concentrations of prolactin (PRL) in serum after a sudden change in photoperiod and daily responsiveness to PRL-releasing and inhibiting factors were investigated in prepubertal Holstein bull calves exposed to different photoperiods. In calves switched from 8-hr light: 16-hr dark to 16-hr light:8-hr dark, there was no observable change in the daily pattern of serum concentrations of PRL after 1, 2, or 4 days. On the other hand, in animals switched from 16-hr light:8-hr dark to 8-hr light:16-hr dark, there was a consistent increase in serum PRL from 33.4 ng/ml on Day 0 to maximum values of 57.3, 62.7, and 78.9 ng/ml between 14 and 18 hr after onset of light on Days 1, 2, and 4, respectively. Thus, absence of light allowed expression of a daily rhythm in serum concentrations of PRL that persisted for at least 4 days after the photoperiod switch. There were no differences in L-dopa inhibition of PRL release in animals exposed to 16-hr light:8-hr dark at 3 or 15 hr after onset of light. However, thyrotropin-releasing hormone-induced release of PRL was greater 3 hr after onset of light (11 hr after onset of dark) compared with release at 9, 15, and 21 hr after onset of light in animals exposed to 16-hr light:8-hr dark, but not in bulls exposed to 8-hr light:16-hr dark. The results provide evidence that the cue for the putative photosensitive period of PRL secretion in cattle may be more closely associated with onset of dark, not onset of light.     

Fertility of Y(TIR).B6 sex-reversal females with XX orthotopic ovarian transplants

When the Y chromosome of Mus musculus domesticus (Y(TIR)) was introduced onto the C57BL/6J (B6) mouse background, testis development was impaired and half of the XY progeny (Y(TIR).B6) developed a female phenotype. Y(TIR).B6 fetal ovaries showed massive death of medullary oocytes and, after birth, produced abnormal levels of steroid hormones, exhibited irregular estrous cycles, and failed to become fertile. In this study we examined whether alterations during perinatal development observed in Y(TIR).B6 ovaries permanently impaired the establishment of the hypothalamus-pituitary-ovary axis (HPOa). B6 fetal and postnatal ovaries at different stages (fetal, infantile, or adult) were transplanted orthotopically (to the ovarian bursa) to either ovariectomized B6 normal females or Y(TIR).B6 sex-reversal females. Percentage of pregnancy, litter size, and capacity to feed pups were recorded. Reciprocally, XY(TIR).B6 ovaries were orthotopically transplanted into B6 females. After crossing with fertile males, several Y(TIR).B6 sex-reversal females with B6 ovarian transplants at all ages became pregnant, had offspring, and fed their pups. On the other hand, none of the B6 female hosts with XY(TIR) ovaries became pregnant. Results demonstrated that Y(TIR).B6 sex-reversal females maintain a functional HPOa and that their failure to reproduce is primarily due to an ovarian defect.     

Regulation of estrous cycle in hysterectomized albino rats by activation of follicular development.

Wistar strain albino rats were hysterectomized and the estrous cycle was compared with sham operated controls. Duration of estrous cycle in hysterectomized rats increased markedly with significant delay in the luteal phase and this was correlated to the inhibited follicular development of ovary. When these rats were treated with PGF2 alpha and PMSG and subjected to physical exercises, the estrous cycle was synchronised and the ovaries of such animals had active follicular development. Thus the deranged operation of sexual cycle in hysterectomized rats was regulated through physical exercises.     

Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis.

The sensitivity of Bacillus subtilis to hydrogen peroxide (oxidative stress) was found to vary with the position of the culture in the growth cycle. The most dramatic change occurred at the stationary phase, when the cells became totally resistant to 10 mM H2O2, in contrast to the loss of 3 to 4 log units of viability when treated at the early log phase. Two of the eight proteins induced by a protective concentration of H2O2 (50 muM) were also induced (in the absence of oxidative stress) on entry into the late log phase of growth. The response of five isogenic spo0 mutants (spo0B, spo0E, spo0F, spo0H, and spo0J) to oxidative stress was identical to that of the wild-type parental strain. In an isogenic spo0A strain, mid-log-phase cells were 100-fold less sensitive to 10 mM H2O2 than was the wild type. Pretreatment with 50 microM H2O2 induced little further protection, suggesting that the response is constitutive in this strain. By comparison of proteins induced by 50 microM H2O2 in the wild-type, spo0A, spo0H, and spo0J strains, four proteins were identified that may be essential for protection against lethal concentrations of H2O2. The presence of multiple copies of the spo0H gene in a spo0A background converted the stress phenotype of the spo0A mutant to that of the wild type but left the sporulation phenotype unaltered.