BRCA-1 Gene Expression and Comparative Proteomic Profile of Primordial Follicles from Young and Adult Buffalo (Bubalus bubalis) Ovaries.

In our previous study, we demonstrated that the repair efficiency of DNA double-strand breaks declines with increasing age in rat primordial follicles. In the present study, we extended our studies to buffalo (Bubalus bubalis) wherein we studied the expression of BRCA-1 related DNA repair genes in primordial follicles of young (12 months-22 months) and adult (72-96 months) buffaloes. The relative expression of selected genes, as determined by RT-PCR, revealed a significant (p < 0.05) decrease in mRNA levels of BRCA1, MRE11, RAD51, ATM, and H2AX in adult primordial follicles as compared to the young. Western blot analysis revealed a significant (p < 0.05) decrease in the expression of phosphorylated protein levels of BRCA1 and H2AX in adult buffalo primordial follicles. The protein expression profile of young and adult buffalo primordial follicles revealed differential expression of proteins involved in mitochondrial function, cell survival and cell metabolism. Similar to reports from aging rodent and human primordial follicles, our findings support the fact that impairment of DNA repair may be an universal mechanism involved in oocyte aging.

Effect of human telomerase reverse transcriptase transfection on differentiation in BeWo choriocarcinoma cells.

Arrest of proliferation is one of the prerequisites for differentiation of cytotrophoblasts into syncytiotrophoblasts, and thus during differentiation telomerase activity, as well as human telomerase reverse transcriptase (hTERT) expression, is down-regulated. Considering this, it is of interest to investigate whether syncytium formation can be delayed by prolonging the expression of telomerase in cytotrophoblasts. BeWo cells were transfected with pLPC-hTERT retroviral vector and the reverse transcription-polymerase chain reaction analysis for hTERT mRNA concentrations in the transfected cells revealed a several-fold increase in hTERT mRNA compared with the cells transfected with empty vector, and this confirmed that the transfection was successful. An increase in the proliferation, as assessed by bromodeoxyuridine incorporation assay, as well as an increase in mRNA and protein concentration of various cyclins and proliferating cell nuclear antigen, was noticed. The effect of hTERT transfection was also assessed after the addition of forskolin to induce differentiation and it was observed that cell-cell fusion was delayed and differentiation did not occur in hTERT-transfected cells. However, the effects seen were only transient as stable transfection was not possible and the cells were undergoing apoptosis after 72 h, which suggested that apart from hTERT other factors might be important for immortalization of BeWo cells.

Current-conveyor-based wide-band current driver for electrical impedance tomography.

OBJECTIVE: In this paper a wide-band integrated current driver for electrical impedance tomography (EIT) is presented. The application is primarily for prostate and breast cancer detection which require the tissue to be interrogated at frequencies up to 10 MHz while achieving low harmonic distortion and high accuracy. APPROACH: The current driver is based on current conveyor architecture and can deliver 1.2 mA of peak to peak ac current between frequencies of 100 Hz-10 MHz. It is fabricated in CMOS 0.18 [Formula: see text]m technology with a power supply of 3.3 V, and occupies a core area of 0.26 [Formula: see text]. MAIN RESULTS: The measured harmonic distortion for a peak current of 1.2 mA is <[Formula: see text] for frequencies less than 100 kHz, and increases to [Formula: see text] at 10 MHz. The measured output impedance of the current driver is 101 k[Formula: see text] at 1 MHz and 19.5 k[Formula: see text] at 10 MHz. SIGNIFICANCE: The circuit is suitable for high frequency active electrode applications.

Gene expression profiling during Forskolin induced differentiation of BeWo cells by differential display RT-PCR.

The differentiation of cytotrophoblasts into syncytiotrophoblasts in the placenta has been employed as a model to investigate stage specific expression as well as regulation of genes during this process. While the cytotrophoblasts are highly invasive and proliferative with relatively less capacity to synthesize pregnancy related proteins, the multinucleated syncytiotrophoblasts are non-proliferative and non-invasive. However, syncytiotrophoblasts are the site of synthesis of a variety of protein, peptide and steroid hormones as well as several growth factors. Both the freshly isolated cytotrophoblasts from human placenta as well as the BeWo cell, a choriocarcinoma cell line model which retain several characteristic of cytotrophoblasts has been employed by us to study regulation of differentiation. In the present study, we have employed the differential display RT-PCR analysis (DD-RT-PCR) to evaluate gene expression changes during Forskolin induced in vitro differentiation of BeWo cells. We have identified several genes which are differentially expressed during differentiation and the differential expression of 10 transcripts was confirmed by Northern blot analysis. Based on the identity of the transcripts an attempt has been made to relate the known function of the gene products, to changes observed during differentiation. Of the several transcripts, one of the transcripts, namely Secretory Leukocyte Protease Inhibitor (SLPI) which is known to have multiple functions was found to increase 15-fold in the syntiotrophoblast.

A simplified laparoscopic ventral hernia repair: the scroll technique.

BACKGROUND: Laparoscopic ventral hernia repair has steadily gained recognition as an alternative to the open approach. However, the procedure can be technically challenging. The authors present their simple scroll technique for laparoscopic ventral hernia repair. METHODS: A total of 174 patients underwent laparoscopic ventral hernia repair using the scroll technique. The technique entails fixation of the rolled mesh to the anterior abdominal wall before it is unfolded. Patient characteristics, operative time, and complications were analyzed and compared with pooled data from the available literature on laparoscopic ventral hernia repair. RESULTS: The mean operative time was comparable with that reported by others (mean, 102 vs. 100 min). The hospital stay was shorter (mean, 1.8 vs. 2.4 h). During a mean follow-up period of 28 months, the recurrence rate was lower than that reported by others (1.7% vs. 4.3%). There were no mortalities and no cases of inadvertent bowel injury. CONCLUSION: The authors' scroll technique for laparoscopic repair is simple, feasible, and reproducible, with a short learning curve and a low recurrence rate.

Expression of functional aromatase in the epididymis: role of androgens and LH in modulation of expression and activity.

The primary source of 17beta-estradiol (E2) in the male is the testis, which expresses the enzyme complex aromatase that is involved in E2 biosynthesis. However, recent evidences suggest that the epididymis is also capable of E2 biosynthesis. Our results demonstrate the presence of cytochrome P450 aromatase (P450(AROM)) and 17beta-hydroxysteroid dehydrogenase I messenger ribonucleic acid (mRNA) in the caput and cauda regions of rat epididymis. The androgenic substrates testosterone and androstenedione could be utilized by the rat epididymal aromatase for E2 biosynthesis as assessed by radioimmunoassay. P450(AROM) expression is transcriptionally regulated in a tissue-specific manner by various factors including androgens and luteinizing hormone (LH). Androgens could positively modulate epididymal P450(AROM) mRNA levels as assessed by castration studies, treatment with flutamide or in vitro incubation of tissue minces with 5 alpha-dihydrotestosterone (DHT). Several extra-gonadal tissues including the epididymis are known to express LH receptors (LHR). Our study revealed a higher level of LHR mRNA expression in the cauda region compared to the caput. Caudal membrane extracts could bind human chorionic gonadotropin (hCG), which resulted in the production of cAMP. Interestingly, hCG could also regulate P450(AROM) mRNA expression in vitro and enhance E2 biosynthesis. Together our results highlight the presence of a functional aromatase in the epididymis that is subject to regulation by LH and androgens.

Differential expression and antibacterial activity of WFDC10A in the monkey epididymis.

The ability of the epididymis to perform its diverse functions stems from its regionalized gene and protein expression patterns. The differences in the gene expression patterns of the caput and cauda regions of the bonnet monkey epididymis were compared using the technique of differential display reverse transcriptase polymerase chain reaction. A transcript showing homology to human whey acidic protein 10 (hWFDC10A) was highly expressed in the monkey caput region. A peptide P2 was designed spanning a region of the monkey WFDC10A (mWFDC10A), which could inhibit the growth of gram-negative bacterial strains of Escherichia coli. P2 could permeabilize the bacterial cell membrane but was unable to permeabilize mammalian cells as evidenced by the lack of hemolysis upon incubation with the peptide. Expression of genes such as mWFDC10A may be essential in providing the first line of defense against microbial infections to the epididymal tract and thus rendering protection to the male gametes sheltered within the epididymis.

FSH, the neglected sibling: evidence for its role in regulation of spermatogenesis and Leydig cell function.

The role of follicle stimulating harmone(FSH) in male reproductive function remains a matter of debate although recent evidences strongly suggest a role despite the controversies that arose following the results obtained with FSH-beta null mice and observations from human FSH receptor mutations. This review summarizes the recent developments of our understanding on the role of FSH in male reproduction. Specifically the results obtained with FSH-beta and FORKO null mice are be discussed in light of our observations employing active and passive neutralization of endogenous FSH in rodents and primates along with other studies. On the basis of results obtained employing a variety of models it can be conclude unequivocally that FSH regulates Leydig cell function and is essential for normal quantitative spermatogenesis.

Neonatal exposure to 17ß-estradiol down-regulates the expression of synaptogenesis related genes in selected brain regions of adult female rats.

AIMS: Administration of estradiol or compounds with estrogenic activity to newborn female rats results in irreversible masculinization as well as defeminization in the brain and the animals exhibit altered reproductive behavior as adults. The cellular and molecular mechanism involved in inducing the irreversible changes is largely unknown. In the present study, we have monitored the changes in the expression of selected synaptogenesis related genes in the sexually dimorphic brain regions such as POA, hypothalamus and pituitary following 17ß-estradiol administration to neonatal female rats. MAIN METHODS: Female Wistar rats which were administered 17ß-estradiol on day 2 and 3 after birth were sacrificed 120days later and the expression levels of genes implicated in synaptogenesis were monitored by semi-quantitative reverse transcription PCR. Since estradiol induced up-regulation of COX-2 in POA is a marker for estradiol induced masculinization as well as defeminization, in the present study only animals in which the increase in expression of COX-2 gene was observed in POA were included in the study. KEY FINDINGS: Down-regulation of genes such as NMDA-2B, NETRIN-1, BDNF, MT-5 MMP and TNF-α was observed in the pre-optic area of neonatally E2 treated female rat brain but not in hypothalamus and pituitary compared to the vehicle- treated controls as assessed by RT-PCR and Western blot analysis. SIGNIFICANCE: Our results suggest a possibility that down-regulation of genes associated with synaptogenesis in POA, may be resulting in disruption of the cyclical regulation of hormone secretion by pituitary the consequence of which could be infertility and altered reproductive behavior.

Effect of calcium depletion on the secretion of newly synthesised human chorionic gonadotropin by first trimester human placenta.

Depletion of calcium in the extracellular medium used to incubate first trimester human placental minces resulted in a significant decrease in the quantity of immunoreactive hCG in the medium and a corresponding increase in the tissue. In contrast, when secretion of newly synthesised hCG was monitored in the absence of calcium by using a radioactive amino acid precursor, a significant increase in the secretion of newly synthesised hCG in the medium was noticed. This was true of secretion of other proteins also as evidenced by the increase in the trichloroacetic acid precipitable radioactivity in the medium in the absence of calcium. These results suggest that newly synthesised hCG is preferentially released over stored hormone in the absence of calcium.

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human.

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein.

Role of oestradiol-17 beta in the regulation of synthesis and secretion of human chorionic gonadotrophin by first trimester human placenta.

Inhibition of aromatase, a key enzyme in the biosynthesis of oestradiol-17 beta, by the addition of 1,4,6-androstatrien-3,17-dione resulted in a significant increase in the levels of immunoreactive human chorionic gonadotrophin (hCG) in the medium and tissue. This increase was partially reversed by the simultaneous addition of oestradiol-17 beta. These effects on the levels of immunoreactive hCG were also reflected by the increased levels of mRNA specific for the alpha and beta subunits of hCG following the addition of the aromatase inhibitor. However, addition of tamoxifen resulted in a drastic decrease in the levels of both the messages. Based on these results, it is suggested that the synthesis of hCG is negatively modulated by oestradiol-17 beta in the human placenta.

Studies on non-specific interference due to serum in the avidin-biotin microELISA for monkey chorionic gonadotropin and a method for its elimination.

A study has been carried out on the non-specific interference due to serum in the avidin biotin microELISA for monkey chorionic gonadotropin. Results suggest that it is not due to any proteolytic activity in the serum, but immunoglobulin or associated factors interfering at the level of antigen-antibody interaction. This interference was eliminated by heating samples at 60 degrees C for 30 min.

Synthesis and biological activity of beta-melanotropins and analogues.

Synthetic [Arg8]-, [Gly10]-, and [Phe12]-beta cl-MSH and beta p-MSH have been prepared by the solid-phase method. Their lipolytic and melanotropic activities have been compared to each other and with those of previously synthesized beta cl-MSH, [formyl-Trp12]-beta cl-MSH, and beta b-MSH. Replacement of Bln8 of beta cl-MSH by Arg causes a greater decrease in melanotropic activity than lipolytic activity. However, when Phe10 of beta cl-MSH is replaced by Gly, both activities are destroyed. Alteration or replacement of Trp12 of beta cl-MSH by formyl-Trp or Phe has little effect on the melanotropic activity while the lipolytic activity is greatly decreased. Replacement of Ser2 of beta b-MSH by Glu induces a three fold increase in melantropic activity but it does not affect the lipolytic activity.

Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model.

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.

A role for calcium in gonadotrophin-releasing hormone (GnRH) stimulated secretion of chorionic gonadotrophin by first trimester human placental minces in vitro.

In vitro studies using first-trimester human placental minces have shown that stimulation of human chorionic gonadotrophin (hCG) secretion by gonadotrophin-releasing hormone (GnRH) is dependent upon the presence of extracellular calcium. Addition of GnRH to first-trimester placental minces in vitro was found to stimulate 45Ca2+ uptake into placental minces, and the process was associated with an increase in immunoreactive hCG in the medium. Addition of GnRH to placental minces preloaded with 45Ca2+ stimulated the efflux of 45Ca2+ within one minute. The calmodulin inhibitors chlorpromazine and trifluoperazine inhibited the basal uptake and efflux of 45Ca2+, suggesting the involvement of calmodulin in the mobilization of calcium in the placenta.

Regulation of protein synthesis in the first trimester human placenta by 17 beta-oestradiol and progesterone.

Addition of 17 beta-oestradiol or progesterone to first trimester human placental explants in vitro resulted in the stimulation of protein synthesis, as seen by autofluorographic analysis of placental tissue and medium proteins. An increase in the incorporation of [35S]methionine into trichloroacetic acid-precipitable proteins was seen, following the addition of 17 beta-oestradiol. Use of aromatase inhibitor to block the synthesis of 17 beta-oestradiol inhibited the protein synthesis and while addition of cyclohexamide blocked both basal- and 17 beta-oestradiol-induced protein synthesis, actinomycin-D blocked only 17 beta-oestradiol induced protein synthesis. Double labelling of placental proteins in the presence and absence of 17 beta-oestradiol also indicated that there is a significant stimulation of protein synthesis by 17 beta-oestradiol. Based on these results it is suggested that oestradiol has a role in regulation of placental protein synthesis.

TGF beta1 induces multiple independent signals to regulate human trophoblastic differentiation: mechanistic insights.

Transforming growth factor-beta 1 (TGF beta1) plays a crucial role in controlling trophoblast growth and invasion. Loss of this key regulatory function provides the pathophysiological basis for several tumors, which are characterized by uncontrolled telomerase activity. We have shown earlier that telomerase activity is negatively regulated during terminal differentiation of human trophoblasts, and that TGF beta1 may be an important factor governing the transcription of human telomerase reverse transcriptase (hTERT) (the catalytic subunit of the telomerase complex) during this process. In the present study, we extend these observations to identify possible functional effectors of TGF beta1-induced loss in telomerase activity during human trophoblastic differentiation. We show that this regulation may involve the suppression of c-Myc and an increased production of Mad1. We also observed a simultaneous increase in the expression of cyclin-dependent-kinase inhibitors, p21, p27, p15 and p16, associated with a loss in expression of Cyclin-A2 and Cyclin-E. Thus, TGF beta1 may induce multiple independent signals to check the proliferative potential of human trophoblastic cells and allow their functional differentiation.

Hormonal regulation of human trophoblast differentiation: a possible role for 17beta-estradiol and GnRH.

We have examined the role of 17beta-estradiol and gonadotropin releasing hormone (GnRH) in the regulation of functional differentiation in human trophoblasts. In contrast to its recognized functions as a proliferation-promoting hormone in a variety of cell types, we found that 17beta-estradiol induced terminal differentiation in human trophoblastic cells, and that this event was estrogen-receptor-mediated. This process involved a loss in expression of Cyclins A2 and E, and a coincident increase in p27(Kip1). The anti-proliferative effects of 17beta-estradiol were annulled by specific transforming growth factor-beta 1 (TGFbeta1)-neutralizing antibody, suggesting that 17beta-estradiol may mediate its growth-inhibitory actions, through TGFbeta1 activity. Following exposure to Buserelin, cultured human trophoblastic cells stopped proliferating and formed functionally mature syncytiotrophoblasts. This differentiation event, that involved a drastic loss in expression of proliferating-cell-nuclear-antigen, could be blocked by Cetrorelix, suggesting the involvement of functional GnRH receptors. Preliminary studies on the characterization of the human placental GnRH receptor, indicate the presence of multiple receptor isoforms across human gestation.

Regulation of telomerase during human placental differentiation: a role for TGFbeta1.

The transient tumor-like attributes of the first-trimester placenta anchor the developing embryo to the uterine wall thus establishing a vital link between the mother and the fetus. Dysregulation of this invasive behavior and/or controlled proliferation of the placenta is associated with abnormal pregnancies. Several of these diseased states also exhibit aberrant telomerase activity, among other pathophysiological manifestations. Considering the strong correlation between telomerase activity and tumorigenesis, it was of interest to see whether the crucial processes of trophoblast proliferation and differentiation were brought about through the modulation of telomerase. Using two in vitro model systems of trophoblast differentiation, we demonstrate here that telomerase activity is negatively regulated during placental differentiation. We further show that this modulation is at the level of transcription of hTERT. We also propose a role for TGF beta1 in regulating telomerase activity in differentiating trophoblasts by down-regulating the expression of hTERT at the transcriptional level.