Cloning, Expression and Hormonal Regulation of Steroidogenic Acute Regulatory Protein Gene in Buffalo Ovary

Malhotra, Nupur,Singh, Dheer,Sharma, M.K. Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.2

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

Vulval premalignant lesions: a review article

Sumedha Gupta,Sana Ahuja,Dheer Singh Kalwaniya,Saritha Shamsunder,Shalu Solanki 대한산부인과학회 2024 Obstetrics & Gynecology Science Vol.67 No.2

Vulvar intraepithelial neoplasia (VIN) is a noninvasive squamous lesion that is a precursor of vulvar squamous cell cancer. Currently, no screening tests are available for detecting VIN, and a biopsy is performed to confirm the clinical diagnosis. Despite sharing many risk factors with cervical intraepithelial neoplasia, the diagnosis of VIN is poses challenges, contributing to its increasing prevalence. This study aimed to analyze the underlying risk factors that contribute to the development of VIN, identify specific populations at risk, and define appropriate treatment approaches. Differentiated VIN (dVIN) and usual VIN (uVIN) are the classifications of VIN. While dVIN is associated with other vulvar inflammatory disorders, such as lichen sclerosis, the more prevalent uVIN is associated with an underlying human papillomavirus infection. Patients with differentiated VIN have an increased risk of developing invasive malignancies. Few effective surveillance or management techniques exist for vulvar intraepithelial neoplasia, a preinvasive neoplasm of the vulva. For suspicious lesions, a thorough examination and focused biopsy are necessary. Depending on the specific needs of each patient, a combination of surgical and medical approaches can be used. Vulvar intraepithelial neoplasia (VIN) is a noninvasive squamous lesion that is a precursor of vulvar squamous cell cancer. Currently, no screening tests are available for detecting VIN, and a biopsy is performed to confirm the clinical diagnosis. Despite sharing many risk factors with cervical intraepithelial neoplasia, the diagnosis of VIN is poses challenges, contributing to its increasing prevalence. This study aimed to analyze the underlying risk factors that contribute to the development of VIN, identify specific populations at risk, and define appropriate treatment approaches. Differentiated VIN (dVIN) and usual VIN (uVIN) are the classifications of VIN. While dVIN is associated with other vulvar inflammatory disorders, such as lichen sclerosis, the more prevalent uVIN is associated with an underlying human papillomavirus infection. Patients with differentiated VIN have an increased risk of developing invasive malignancies. Few effective surveillance or management techniques exist for vulvar intraepithelial neoplasia, a preinvasive neoplasm of the vulva. For suspicious lesions, a thorough examination and focused biopsy are necessary. Depending on the specific needs of each patient, a combination of surgical and medical approaches can be used.

The evolving landscape of immunohistochemistry in cervical and uterine carcinoma in gynecologic oncology: current status and future directions

Sumedha Gupta,Sana Ahuja,Dheer Singh Kalwaniya 대한산부인과학회 2024 Obstetrics & Gynecology Science Vol.67 No.5

Immunohistochemistry (IHC) has become an indispensable tool in routine gynecological pathology, particularly with the advancements in molecular understanding and histological classification of gynecological cancers. This evolution has led to new immunostainings for diagnostic and classification purposes. This review describes the diagnostic utility of IHC in gynecological neoplasms, drawing insights from literature reviews, personal experiences, and research findings. It delves into the application of IHC in resolving morphologically equivocal cases, emphasizing its role in achieving an accurate diagnosis. The selection of appropriate immunomarkers for common scenarios encountered in gynecological pathology aids pathologists in navigating complex cases. Specifically, we focus on cervical and endometrial malignancies, elucidating the molecular rationale behind the use of specific immunohistochemical markers. An updated overview of essential immunohistochemical markers provides knowledge for precise diagnosis and classification of gynecological cancers. This review serves as a valuable resource for clinicians and researchers involved in the management and study of gynecological malignancies, facilitating improved patient care and outcomes.

An Insight of Nanomaterials in Tissue Engineering from Fabrication to Applications

Sharma Ritika,Kumar Sanjeev,Bhawna,Gupta Akanksha,Dheer Neelu,Jain Pallavi,Singh Prashant,Kumar Vinod 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.5

Tissue engineering is a research domain that deals with the growth of various kinds of tissues with the help of synthetic composites. With the culmination of nanotechnology and bioengineering, tissue engineering has emerged as an exciting domain. Recent literature describes its various applications in biomedical and biological sciences, such as facilitating the growth of tissue and organs, gene delivery, biosensor-based detection, etc. It deals with the development of biomimetics to repair, restore, maintain and amplify or strengthen several biological functions at the level of tissue and organs. Herein, the synthesis of nanocomposites based on polymers, along with their classification as conductive hydrogels and bioscaffolds, is comprehensively discussed. Furthermore, their implementation in numerous tissue engineering and regenerative medicine applications is also described. The limitations of tissue engineering are also discussed here. The present review highlights and summarizes the latest progress in the tissue engineering domain directed at functionalized nanomaterials.

Validation of Gene Silencing Using RNA Interference in Buffalo Granulosa Cells

Monga, Rachna,Datta, Tirtha Kumar,Singh, Dheer Asian Australasian Association of Animal Productio 2011 Animal Bioscience Vol.24 No.11

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

Methyl Coenzyme M Reductase (mcrA) Gene Based Phylogenetic Analysis of Methanogens Population in Murrah Buffaloes (Bubalus bubalis)

Prem Prashant Chaudhary,Sunil Kumar Sirohi,Dheer Singh,Jyoti Saxena 한국미생물학회 2011 The journal of microbiology Vol.49 No.4

The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant methanogenic communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.

Effect of Follicular Fluid Proteins and Gonadotropins on Progesterone Secretion by Buffalo Granulosa Cells In vitro

Vinze, Mukesh,Sharma, M.K.,Singh, Dheer Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.11

In the mammalian ovary the follicular fluid contains proteins and peptides which play an important role in growth, development and maturation of oocytes. The gonadotropins and some other factors work synergistically and regulate ovarian functions. In the present study the effect of follicular fluid proteins (FFP) and gonadotropins on progesterone secretion by granulosa cells (GC) from buffalo ovary, was investigated during culture. The follicular fluid was collected from small (<5 mm), and medium (5-8 mm) follicles obtained from buffalo ovaries. The follicular fluid from medium follicles was fractionated with ammonium sulphate at 80% saturation. The precipitated protein fraction was further resolved in to minor (peaks I, III) and major (peak II) proteins using gel filtration (Sephadex G-200). The FFP from small follicles and major FFP (peak II) at a dose of 200 $\mu$g/well, significantly stimulated progesterone secretion by pooled GC (3${\times}10^{5}$ cells/2 ml medium/well). The minor FFP did not show any stimulatory effect. There was a significant increase in progesterone secretion by pooled GC in presence of FFP and LH (10 ng/well), however, FSH (20 ng/well) with FFP exhibited an inhibitory effect. The major FFP and gonadotropins were also studied for their effect on progesterone production by GC isolated from medium and large size follicles. The GC from medium follicles were more responsive to FSH and FFP whereas GC from large follicles exhibited enhanced progesterone secretion with LH and FFP. These results indicated that FFP have their own stimulatory effect and also act synergistically with gonadotropins. The significantly different response shown by GC, for steroid hormone secretion, is based on their stage of growth and differentiation. The purification and characterization of such steroidogenic proteins may help in elucidating their role in growth and differentiation of granulosa cells.