N H Balasinor

NIRRH, Neuroendocrinology, Faculty Member

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Papers by N H Balasinor

Genetics: I-50: Embryo Loss Due to Epigenetic Anomalies in the Male Germ Line: Role of Estrogen

Endocrine control of epigenetic mechanisms in male reproduction

Hormone Molecular Biology and Clinical Investigation, 2016

Endocrine control of reproduction is very well known and has been echoed by many research groups.... more Endocrine control of reproduction is very well known and has been echoed by many research groups. However, recent developments point to the ability of toxic endocrine disrupting chemicals (EDC) to alter epigenetic information of the gametes which gets transferred to the developing embryo and affects the immediate reproductive outcome or even persists transgenerationally. These epigenetic aberrations contribute to the ensuing pathophysiology of reproductive disorders. Investigations of the female in cases of poor reproductive outcome have been the main strategy towards diagnosis. However, despite the male partner contributing half of his genome to the progeny, thorough investigations in the male have been ignored. Environmental pollutants are all pervading and are encountered in our day-to-day life. Many of these pollutants have potential to disrupt the endocrine system. Here, we discuss how the male gametes (spermatozoa) are susceptible to a myriad of epigenetic insults inflicted by...

Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals

Fertility and Sterility, 2012

To study H19 ICR methylation levels in association with sperm parameters routinely analyzed in id... more To study H19 ICR methylation levels in association with sperm parameters routinely analyzed in idiopathic recurrent spontaneous miscarriage cases. Design: Case-control study. Setting: Academic research setting. Patient(s): Male partners of couples with a history of idiopathic recurrent spontaneous miscarriage (RSM group) and male partners of couples with proven fertility (control group). Intervention(s): None. Main Outcome Measure(s): Paternal age, sperm concentration, motility, chromatin compaction status, morphology, and H19 ICR methylation were assessed in control and idiopathic RSM group participants. Result(s): Paternal age and basic semen parameters analyzed did not show any significant difference between the two groups; however H19 ICR methylation levels were reduced significantly in the idiopathic RSM group compared with the control group. Conclusion(s): Significant reduction in the H19 ICR methylation without significant difference in the sperm parameters demonstrates aberrant imprinting to be associated with idiopathic RSM.

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Methylation status of imprinted genes DLK1-GTL2, MEST (PEG1), ZAC (PLAGL1), and LINE-1 elements in spermatozoa of normozoospermic men, unlike H19 imprinting control regions, is not associated with idiopathic recurrent spontaneous miscarriages

Fertility and Sterility, 2013

Objective: To study methylation aberrations in spermatozoa at developmentally important imprinted... more Objective: To study methylation aberrations in spermatozoa at developmentally important imprinted regions to ascertain their role in early embryo loss in idiopathic recurrent spontaneous miscarriages (RSM). Design: Case-control study. Setting: Academic research setting at National Institute for Research in Reproductive Health, Parel, Mumbai. Patient(s): Male partners of couples with a history of RSM and male partners of couples with proven fertility (control group). Intervention(s): None. Main Outcome Measure(s): DNA methylation levels at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) by Epityper Massarray and global methylation levels as measured by LINE-1 methylation and anti-5-methyl cytosine antibody in spermatozoa of 23 men in control group and 23 men in RSM group. Result(s): We did not observe any aberration in the total methylation levels in any of the imprinted genes or global methylation analyzed. Conclusion(s): Our results indicate that paternal methylation aberrations at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) and global methylation levels are not associated with idiopathic RSM and may not be good epigenetic markers (unlike the H-19 imprinting control region) for diagnosis of idiopathic RSM.

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Effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 locus-specific DNA methylation in rat spermatozoa and its association with embryo loss

Fertility and Sterility, 2009

Objective: To determine the effect of tamoxifen treatment on global and insulin-like growth facto... more Objective: To determine the effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 imprinting control region (Igf2-H19 ICR)-specific DNA methylation in rat spermatozoa and analyze its association with postimplantation loss. Design: Experimental prospective study. Setting: Animal research and academic research facility. Subject(s): Male and female 75-day-old Holtzman rats. Intervention(s): Global and Igf2-H19 ICR-specific DNA methylation was analyzed in an epididymal sperm sample in control and tamoxifen-treated rats at a dose of 0.4 mg tamoxifen/kg/day. DNA methylation status was correlated to postimplantation loss in females mated with tamoxifen-treated males. Main Outcome Measure(s): Global sperm DNA methylation level, methylation status of Igf2-H19 ICR in sperm, postimplantation loss. Result(s): Tamoxifen treatment significantly reduced methylation at Igf2-H19 ICR in epididymal sperm. However, the global methylation level was not altered. A mating experiment confirmed a significant increase in postimplantation loss upon tamoxifen treatment and showed significant correlation with methylation at Igf2-H19 ICR. Conclusion(s): Reduced DNA methylation at Igf2-H19 ICR in rat spermatozoa upon tamoxifen treatment indicated a role of estrogen-associated signaling in the acquisition of paternal-specific imprints during spermatogenesis. In addition, association between DNA methylation and postimplantation loss suggests that errors in paternal imprints at Igf2-H19 ICR could affect embryo development.

format_quoteTamoxifen treatment significantly increased postimplantation loss from 2.11% in control to 8.9% in treated male rats (P<0.05).format_quote

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Endocrine regulation of sperm release

Reproduction, Fertility and Development, 2018

Spermiation (sperm release) is the culmination of a spermatid’s journey in the seminiferous epith... more Spermiation (sperm release) is the culmination of a spermatid’s journey in the seminiferous epithelium. After a long association with the Sertoli cell, spermatids have to finally ‘let go’ of the support from Sertoli cells in order to be transported to the epididymis. Spermiation is a multistep process characterised by removal of excess spermatid cytoplasm, recycling of junctional adhesion molecules by endocytosis, extensive cytoskeletal remodelling and final spermatid disengagement. Successful execution of all these events requires coordinated regulation by endocrine and paracrine factors. This review focuses on the endocrine regulation of spermiation. With the aim of delineating how hormones control the various aspects of spermiation, this review provides an analysis of recent advances in research on the hormonal control of molecules associated with the spermiation machinery. Because spermiation is one of the most sensitive phases of spermatogenesis to variations in hormone levels,...

format_quoteBlocking androgen action resulted in 90% of spermatids failing to spermiate, revealing its critical role in spermatid disengagement.format_quote

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Direct regulation of genes involved in sperm release by estrogen and androgen through their receptors and coregulators

The Journal of Steroid Biochemistry and Molecular Biology, 2017

Highlights • Actin remodeling-related genes (Arpc1b, Evl) have a functional estrogen response ele... more Highlights • Actin remodeling-related genes (Arpc1b, Evl) have a functional estrogen response element • Endocytosis-related genes (Picalm, Eea1, Stx5a) have a functional androgen response element • Estrogen regulates Arpc1b and Evl expression through ER beta via NCoR1 and Src1, respectively • Androgen regulates Picalm and Eea1 expression via NCoR1 and Stx5a expression via Src1 • Estrogen regulates actin remodeling, while androgen regulates endocytosis during sperm release A c c e p t e d M a n u s c r i p t 2 Title: Direct regulation of genes involved in sperm release by estrogen and androgen through their

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Biochimica et biophysica acta, Aug 23, 2016

Actin remodeling is a vital process for signaling, movement and survival in all cells. In the tes... more Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Kno...

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Differential roles of estrogen receptors, ESR1 and ESR2, in adult rat spermatogenesis

Molecular and cellular endocrinology, Jun 19, 2016

Estrogens, through their receptors, play an important role in regulation of spermatogenesis. Howe... more Estrogens, through their receptors, play an important role in regulation of spermatogenesis. However, the precise role of the estrogen receptors (ESR1 and ESR2) has been difficult to determine as in vivo estradiol treatment would signal through both the ESRs. Hence we had developed in vivo selective ESR agonist administration models in adult male rats to decipher the individual roles of the ESRs. Treatment with both ESR1 and ESR2 agonists decreased sperm counts after 60 days of treatment. The present study aimed to delineate the precise causes of decreased sperm counts following treatment with the two ESR agonists. Treatment with ESR1 agonist causes an arrest in differentiation of round spermatids into elongated spermatids, mainly due to down-regulation of genes involved in spermiogenesis. ESR2 agonist administration reduces sperm counts due to spermiation failure and spermatocyte apoptosis. Spermiation failure observed is due to defects in tubulobulbar complex formation because of ...

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Localization of proteins involved in endocytosis at tubulobulbar complexes in rat testes

Advances in Reproductive Sciences, 2014

Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermati... more Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.

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Estrogen and androgen regulate actin-remodeling and endocytosis-related genes during rat spermiation

Molecular and cellular endocrinology, Jan 28, 2015

Spermiation, the sperm release process, is imperative to male fertility and reproduction. Morphol... more Spermiation, the sperm release process, is imperative to male fertility and reproduction. Morphologically, it is characterized by removal of atypical adherens junctions called ectoplasmic specializations, and formation of transient endocytic devices called tubulobulbar complexes requiring cytoskeleton remodeling and recruitment of proteins needed for endocytosis. Earlier, estrogen administration to adult male rats was seen to cause spermiation failure due to disruption of tubulobulbar complexes. This was accompanied by reduction in intratesticular testosterone levels and increase in intratesticular estrogen along with deregulation of genes involved in cytoskeleton remodeling (Arpc1b, Evl and Capg) and endocytosis (Picalm, Eea1 and Stx5a). In the present study, we aim to understand the role of estrogen and androgen in regulating these genes independently using seminiferous tubule culture system treated with estrogen, androgen or agonists and antagonists of estrogen receptors. We find...

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Delineating the regulation of estrogen and androgen receptor expression by sex steroids during rat spermatogenesis

The Journal of steroid biochemistry and molecular biology, 2018

Estrogen receptors (ERα and β) and androgen receptor (AR) regulate various critical processes dur... more Estrogen receptors (ERα and β) and androgen receptor (AR) regulate various critical processes during spermatogenesis. Since spermatogenesis is very sensitive to hormonal stimuli and perturbations, it is important to understand the regulation of expression of these receptors by sex steroid hormones. Although many studies have reported deregulation of steroid receptors on endocrine disruption, there is no consensus on the regulation of their expression by steroid hormones during spermatogenesis, and a lack of clear understanding of the mechanism of regulation. Here, we evaluated the receptor expressions in a well-established exogenous estradiol administration model. We then investigated the mechanisms by which the individual receptors regulate their expression by binding to the respective hormone response elements upstream of these receptor genes. By further employing in vitro and in vivo models of ER and AR stimulation or antagonism, we delineated their regulation in a receptor subty...

format_quoteERα stimulates Esr1 expression, while ERβ suppresses it, indicating complex interaction dynamics.format_quote

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Proteomics in reproductive biology: Beacon for unraveling the molecular complexities

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2013

Proteomics, an interface of rapidly evolving advances in physics and biology, is rapidly developi... more Proteomics, an interface of rapidly evolving advances in physics and biology, is rapidly developing and expanding its 26 potential applications to molecular and cellular biology. Application of proteomics tools has contributed towards 27 identification of relevant protein biomarkers that can potentially change the strategies for early diagnosis and treat-28 ment of several diseases. The emergence of powerful mass spectrometry-based proteomics technique has added a 29 new dimension to the field of medical research in liver, heart diseases and certain forms of cancer. Most proteomics 30 tools are also being used to study physiological and pathological events related to reproductive biology. There have 31 been attempts to generate the proteomes of testes, sperm, seminal fluid, epididymis, oocyte, and endometrium from 32 reproductive disease patients. Here, we have reviewed proteomics based investigations in humans over the last 33 decade, which focus on delineating the mechanism underlying various reproductive events such as spermatogene-34 sis, oogenesis, endometriosis, polycystic ovary syndrome, embryo development. The challenge is to harness new 35 technologies like 2-DE, DIGE, MALDI-MS, SELDI-MS, MUDPIT, LC-MS etc., to a greater extent to develop widely 36 applicable clinical tools in understanding molecular aspects of reproduction both in health and disease.

format_quoteFour candidate biomarkers for non-obstructive azoospermia were identified, enhancing male infertility diagnostics.format_quote

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Estradiol: A Steroid with Multiple Facets

Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme, Jan 22, 2018

Seventy-five glorious years have passed since estradiol was discovered by Edward Doisy. From disc... more Seventy-five glorious years have passed since estradiol was discovered by Edward Doisy. From discovery in the ovaries to delineation of diverse physiological effects, research on estrogens has covered a lot of ground. Estrogen receptors that mediate estrogenic effects, have been detected not only in reproductive organs, but also in other body organs. Estrogen receptors function either as conventional transcription factors or as rapid signal transducers. These different modes of action are opted by estrogens to elicit an array of reproductive and non-reproductive functions. It is well established that estrogens promote cell proliferation in various tissues and hence are also linked to carcinogenesis. Anti-estrogens are being used as adjunct therapies for cancers since several years. On the other hand, estrogen-based strategies are used to alleviate adverse effects of menopause. Apart from estrogens synthesized in various organs, exposure to environmental estrogens can also impact phy...

Male Infertility: An Epigenetic Perspective

Male Infertility: Understanding, Causes and Treatment, 2017

Besides known genetic and environmental factors, research over the last two decades has shed ligh... more Besides known genetic and environmental factors, research over the last two decades has shed light on several epigenetic mechanisms and their association with male infertility. The male germ line undergoes extensive epigenetic remodeling throughout fetal to adult life and is thus susceptible to environmental factors that can affect fertility. During fetal life, the primordial germ cells undergo removal of epigenetic marks (demethylation) followed by re-establishment of these marks according to the sex of the fetus, at the time of gonadal differentiation. Extensive programming of the epigenome occurs during the various phases of spermatogenesis, i.e., mitosis, meiosis, and spermiogenesis, leading to haploid-condensed spermatozoa with protamines as the major nucleoproteins. Shortly after fertilization, the sperm chromatin decondenses and the protamines are replaced by histones. The male pronucleus undergoes active demethylation. One such epigenetic phenomenon, genomic imprinting resulting in monoallelic expression of genes depending on the parent of origin, is involved in early embryogenesis. Aberrant methylation pattern of imprinting control region (ICR) of imprinted genes in the spermatozoa is associated with altered sperm morphology, count, and motility. This chapter provides a comprehensive overview of the epigenetic changes affecting spermatogenesis and male fertility.

Aberrant methylation of multiple imprinted genes in embryos of tamoxifen-treated male rats

Reproduction, 2013

Genomic imprinting is an epigenetic phenomenon known to regulate fetal growth and development. St... more Genomic imprinting is an epigenetic phenomenon known to regulate fetal growth and development. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased postimplantation loss around mid gestation. Further studies demonstrated the aberrant expression of transcripts of several imprinted genes in the resorbing embryos at days 11 and 13 of gestation including IGF2. In addition, decreased methylation at theIgf2–H19imprint control region was observed in spermatozoa and in resorbing embryos sired by tamoxifen-treated males. In this study, methylation analysis of the imprinted genes, which were found to be differentially expressed, was done using EpiTYPER in the spermatozoa of tamoxifen-treated rats and in postimplantation embryos sired by tamoxifen-treated rats. Differentially methylated regions (DMRs) for most imprinted genes have not been identified in the rats. Hence, initial experiments were performed to identify the putative DMRs in the g...

format_quoteTamoxifen-treated male rats showed a significant increase in postimplantation loss, 10±2% compared to 3±0.68% in controls (P<0.05).format_quote

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DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss

Human Reproduction

STUDY QUESTION What is the sperm DNA methylation status of imprinted genes in male partners from ... more STUDY QUESTION What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY RPL is defined as loss of two or more pregnancies, affecting 1–2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative ...

Aberrant expression of imprinted genes in post-implantation rat embryos

Life Sciences, 2011

Imprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstra... more Imprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 (Igf2), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males.Gene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected p...

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Disruption of Tubulobulbar Complex by High Intratesticular Estrogens Leading to Failed Spermiation

Endocrinology, Feb 3, 2013

Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into th... more Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into the lumen of the seminiferous tubules. Morphologically, it involves a series of events, namely removal of excess spermatid cytoplasm, removal of ectoplasmic specialization, formation of tubulobulbar complex, and final disengagement of the spermatid from the Sertoli cell. Previous studies in our laboratory have shown that administration of 17␤-estradiol at a dose of 100 g/kg body weight for 10 d resulted in failure of spermiation. This was accompanied by a suppression of FSH and intratesticular testosterone with a concomitant rise in intratesticular 17␤-estradiol. The present study was undertaken to determine the cause of failure and subsequently the molecular events in spermiation. Electron microscopic and confocal studies revealed an absence of tubulobulbar complex in step 19 spermatids after estradiol treatment, highlighting the significance of these structures in spermiation. It was further observed that treatment affected the Sertoli cell cytoskeleton and Arp2/3 complex that is critical for de novo polymerization of actin during tubulobulbar complex formation. In conclusion, the present study reports the role of 17␤-estradiol in inhibiting the formation of tubulobulbar complex, which could be one of the mechanism by which environmental estrogens influence male fertility.

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Effect of estrogen receptor subtype specific ligands on fertility in adult male rats

The Journal of endocrinology, Jan 13, 2015

Maintenance of normal male fertility relies on the process of spermatogenesis which is under comp... more Maintenance of normal male fertility relies on the process of spermatogenesis which is under complex endocrine control of gonadotropin and steroid hormones. Although testosterone is the primary sex steroid in males, estrogen is locally produced in the testis and plays a very crucial role in male fertility. This is evident from presence of both the estrogen receptors (ER) α and β in the testis and their absence, as in case of knockout mice models, leads to sterility. The present study was undertaken to understand individual roles of the two ERs in spermatogenesis and their direct contribution towards the maintenance of male fertility using receptor subtype specific ligands. Administration of ERα and β agonists to adult male rats for 60 days results in a significant decrease in fertility mainly due to an increase in pre- and post-implantation loss and a concomitant decrease in litter size and sperm counts. Our results indicate that ERα is mainly involved in negative feedback regulatio...

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