The effect of IBMX and hormones on gene expression by rat Sertoli cells

Accepted Manuscript The Effect of IBMX and hormones on gene expression by rat Sertoli cells Indrashis Bhattacharya, Mukkesh Gautam, Subeer S. Majumdar PII:

S2214-420X(14)00005-9

DOI:

10.1016/j.jrhm.2014.12.001

Reference:

JRHM 4

To appear in:

International Journal of Pediatrics and Adolescent Medicine

Received Date: 8 November 2014 Revised Date:

6 December 2014

Accepted Date: 12 December 2014

Please cite this article as: Bhattacharya I, Gautam M, Majumdar SS, The Effect of IBMX and hormones on gene expression by rat Sertoli cells, International Journal of Pediatrics and Adolescent Medicine (2015), doi: 10.1016/j.jrhm.2014.12.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The Effect of IBMX and hormones on gene expression by rat Sertoli cells Indrashis Bhattacharya 1,a, Mukkesh Gautam 1,b, Subeer S Majumdar 1, c 1= Cellular Endocrinology Lab, National Institute of Immunology, Aruna Asaf Ali Marg, New

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Delhi- 110067.

a= Present address: Department of Zoology, Hemwati Nandan Bahuguna Garhwal University,

b=Present Address:

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Srinagar, India.

The Ken & Ruth Davee Department of Neurology, Northwestern

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University, USA.

c= To whom correspondence should be addressed. E Mail: [email protected]

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Corresponding Author: Subeer S Majumdar

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The Effect of IBMX and hormones on gene expression by rat Sertoli cells

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Abstract

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Background : Sertoli cells (Sc) regulate spermatogenesis under the control of FSH and

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testosterone (T). Functional maturation of Sc for supporting the spermatogenic onset during

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pubertal development is prerequisite for male fertility. However, the effect of hormone driven

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maturational changes in Sc is not well known.

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Objectives and experimental model: In this present study we have compared hormone induced

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gene expression of immature and mature Sc isolated from neonatal (9-days old) and prepubertal

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(18-days-old) rat testes, respectively, to investigate the developmental difference of hormone

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responsiveness of Sc during postnatal maturation as well as influence of 3-isobutyl-1-

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methylxanthine (IBMX), a nonspecific inhibitor of phosphodiesterase in primary culture of Sc .

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Results and conclusion: Our results suggested that FSH responsiveness of Sc obtained from 18-

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days-old rats were more prominent in terms of augmentation of lactate, cAMP and gene

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transcription as compared to Sc from 9-days of age. Our result also indicated that although the

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use of IBMX in primary culture of Sc generates a better readout in terms of FSH induced cAMP

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response, the presence of such pharmacological agent mellows down FSH stimulated gene

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expression profile. Our data indicated further that immature Sc are capable of differentiating in

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vitro if cultured with continuous supplementation of FSH and T (in combination).

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together, we also concluded that for accurate evaluation of the modulation of gene expression by

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hormones, use of IBMX should be avoided in primary cultures of Sc.

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Introduction

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Testicular Sertoli cells (Sc) is the principle site of FSH and testosterone (T) action to regulate

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spermatogenesis1,2. Sc develop specialized Sc-Sc junctions inside the seminiferous tubules to

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establish the blood-testes barrier (BTB) and spermatogenesis takes place inside this specialized

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niche provided exclusively by Sc. The synergistic effect of FSH and T promotes the secretion of

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various growth factors and metabolites by Sc to support the initiation and maintenance of male

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germ cell (Gc) differentiation1,2. A paradoxical situation exists in neonatal rodents3 and infant

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primates4, where despite sufficient levels of hormones (both FSH and T) Sc fails to support the

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spermatogenic onset. Transplantation of Gc isolated from neonatal testes5 to a prepubertal wild

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type background induces appropriate differentiation of the doner Gc towards sperm production.

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This observation confirmed that the failure of differentiation in Gc of neonatal testes stems from

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the defective somatic counterpart of the seminiferous tubule (mainly Sc). We have recently

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reported that in rats, the robust division and differentiation of Gc initiate within the seminiferous

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tubules at around 12 days of postnatal age without any appreciable rise in the circulatory levels

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of FSH and T3 . Therefore, it is reasonable to assume that rat Sc undergo substantial

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developmental changes6 during prepubertal development to incur necessary hormonal

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responsiveness to promote Gc differentiation. Therefore, such developmental changes in Sc

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become prerequisite for male fertility7.

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Functions of Sc are mostly established by in vitro cell culture studies of 18 days old rats8.

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However, hormone mediated maturational changes in Sc enabling them to support

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spermatogenesis, is extremely limited3,4 . A comparative study of gene expression in Sc from

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spermatogenically inactive (9-days-old) and active (18-days-old) testes may reveal the effect of

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the changes in Sc responsible for the induction of Gc differentiation. Here, we here have

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demonstrated the role of hormonal supplementations on in vitro Sc maturation. Additionally we

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also investigated the utility of the use of 3-isobutyl-1-methylxanthine (IBMX), a nonspecific

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inhibitor of phosphodiesterase (PDE) in primary culture of Sc obtained from neonatal and

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prepubertal rats.

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Material and Methods

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Animals and reagents

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Wistar rats (Rattus norvegicus) were obtained from the Small Animal Facility of the National

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Institute of Immunology (New Delhi, India). All animals were housed and used as per the

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national guidelines provided by the Committee for the Purpose of Control and Supervision of

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Experiments on Animals. Protocols for the experiments were approved by the Institutional

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Animal Ethics Committee. Ovine (o)FSH, and anti-cAMP antibody were obtained from National

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Hormone and Pituitary Program (NHPP), National Institutes of Health (NIH; Torrance, CA). All

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other reagents, unless stated otherwise, were procured from Sigma Chemical (St. Louis, MO).

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Isolation of Sc

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Testes were obtained from rats of various postnatal ages 9 (neonatal) or 18 days old

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(prepubertal). Sc were isolated using a sequential enzymatic digestion that has been previously

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described in detail by us3. Since germ cell (Gc) membranes are more fragile than that of the Sc,

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in order to remove the contaminated Gc from the isolated Sc clusters, the clusters were exposed

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to a hypotonic shock by 20 mM Tris- HCl (pH 7.4) for 3–5 min, which is selectively known to

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destroy germ cells sparing Sc3. After two washes with buffer, the cells were treated with Trizol

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before storage at -80°C for subsequent RNA extraction. Data generated from freshly isolated Sc

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were considered as closer to in vivo as it took around 3hr from obtaining the testes from the rats

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to purification of the Sc.

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Long-term culture

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On the first day, i.e. day 0 of culture, isolated Sc clusters were counted under an inverted phase

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contrast microscope (Nikon, DIAPHOT 300, under 20 magnification) and were seeded at a

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density of 0.25 X105 cell clusters per well per ml for 9-day-old rats, and 0.5 X105 cell clusters

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per well per ml for 18-day-old rats, as previously reported by us3. Cultures were continued in

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DMEM-nutrient mixture F-12 Ham (DMEM-F12 HAM) containing 1% FCS for 24 h in a

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humidified 5% CO2 incubator at 34°C. Next day, cells were washed with pre-warmed medium

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(DMEM-F12 HAM) and cultured further in serum replacement growth factor medium (GF

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medium) containing 5 µg/ml sodium selenite, 10 µg/ml insulin, 5µg/ml transferrin, and 2.5 ng/ml

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epidermal growth factor. On day 2 of culture, residual Gc that remain round (while Sc cytoplasm

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expand), if any, were removed by hypotonic shock by incubating Sc with 20 mM Tris·HCl (pH

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7.4) for 3–5 min at 34°C3. Sc were then washed twice to remove dead Gc, and the culture was

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continued further in GF medium. On day 3 of culture, one portion of Sc of each age group was

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treated with Trizol and stored at -80°C for RNA extraction (0 h), and the rest were given various

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treatments. A detailed experimental work plan is given in Table 1.

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Purity of culture

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Cells were cultured on coverslips for 4 days (in GF medium without hormone supplementation)

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and stained with vimentin antibody (Abcam, USA, Ab8978) to detect Sc as described by us in

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detail previously3. PTc and Lc contamination in the culture was identified by determining the

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alkaline phosphatase and the 3β-HSD activity, respectively as described by us earlier3.

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In vitro treatments

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Lactate produced by Sc FSH treatment

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On day 3 of culture, Sc were treated with i) GF media alone ii) GF media containing o-FSH (50

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ng/ml) in presence or absence of 3-isobutyl-1-methylxanthine (IBMX ,10–4 M) for 24 hrs and the

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Sc exposed media were collected and were stored in -80°C to measure the lactate produced and

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secreted by Sc. Cells from each well were dislodged by Trypsin EDTA and were counted using

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hemocytometer.

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Production of cAMP and gene expression under the influence of FSH

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On day 3 of culture, Sc were treated with i) GF media alone ii) GF media containing 50 ng/ml o-

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FSH (obtained from National Hormone and Pituitary Program (NHPP), National Institutes of

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Health; Torrance, CA,) in presence or absence of IBMX (10–4 M) for 2hrs, 4hrs, 8hrs, 12hrs and

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24hrs and the Sc exposed media were collected and stored in -80°C for evaluating the cAMP

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produced and secreted by Sc. Cells from each well were dislodged by Trypsin EDTA and were

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counted using hemocytometer and then saved in Trizol for future RNA extraction.

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Augmentation of hormone responsive genes

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A faction from each well of above treatments was used for cell counting using hemocytometer

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and rest of the Sc were washed, pelleted and treated with Trizol and stored in -80°C for future

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mRNA extraction to evaluate status of gene expression . One fraction of freshly isolated Sc from

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the testes of rats of different age groups were washed and treated with Trizol before storing in -

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80°C for future mRNA extraction. Rest of fraction of the isolated Sc was cultured for 4 days in

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presence or absence of o-FSH and T (FT). After every 24hr, the culture (for each age group) was

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terminated i.e. day 1, day 2 and finally day 3, by treating the cells with Trizol and stored at -80°C

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for future mRNA extraction. For each day of culture, there were both FT untreated (control) and

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FT treated groups.

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Lactate Assay

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Lactate present in the culture media was measured as described in the lactate assay kit (Sigma,

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USA) with some modifications. Briefly, the reaction mixture comprised of 10mg NAD+, 2ml

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glycine buffer and 100U lactate dehydrogenase. Standard curve for lactate was obtained in the

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range of 0.66 to 6.34 µg. The reaction mixture (100µl/500µl reaction volume) was added to the

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lactate standards and samples, incubated at 370C water bath for 15min and the absorbance was

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measured at 340 nm.

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Analyses of the mRNA Expression of the Hormones Regulated Genes by Semi quantitative RT-

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PCR

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Total RNA was isolated from the Trizol treated samples and the purity of the RNA was

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determined by spectrophotometer. RNA having 1.8 or higher value of the 260/280 ratio was used

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for analysis. Total 1µg of RNA from each treatment group was first reverse transcribed using

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Reverse Transcription (RT) System (Promega Corp, USA) with AMV reverse transcriptase and

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oligo (dT)15 for the single-strand cDNA synthesis. Subsequent PCR reactions (10µl reaction

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volume) were carried out using 1µl of the RT reaction as template for checking the expression

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profile of each gene. For each gene number of PCR cycles were standardized to detect an

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acceptable expression level to confirm the findings. The list of genes (both target genes and

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housekeeping gene cyclophilin A) along with primer sequences, annealing temperature (Tm) and

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PCR product sizes are given in Table-2.

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Data representation and statistical analysis

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One treatment group comprised three wells within one culture set. At least three such sets of

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cultures for each age group (performed on different calendar dates) were used to interpret the

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data. Testes from about 20–25 and 6–10 male rats were pooled for 9- and 18-day-old rat Sc

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cultures, respectively. RT-PCR images were captured and analyzed for densitometry by Bio-Rad

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Gel Documentation system. One-way ANOVA followed by Dunnett’s test using the InStat v. 3.0

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statistical program (Graphpad Software, San Diego, CA) was used for statistical analyses of the

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data.

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Results

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Viability and Cytochemical evaluation of the purity of cultured Sc

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To check the viability and purity of Sc culture, trypan blue, vimentin staining (Sc specific) and

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alkaline phosphatase activity (for PTc contamination) were performed respectively. Viability of

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Sc on day 3 of culture was found to be > 98% and contamination of PTc was < 2% in both the

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age groups of rats (data not shown). Imaging analyses confirmed that the isolation procedure

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resulted in highly enriched Sc fractions. Approximately 95% of the cells stained with the Sc

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specific marker vimentin (in both the age groups, Fig: 1. A-H).

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Use of IBMX in detecting hormone (FSH) response –

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Estimation of Lactate

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FSH did not induce a significant (P< 0.05%) rise in lactate production in Sc isolated and cultured

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from 9-days-old rats (Fig: 2.A). However, FSH augmented lactate production in Sc isolated and

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cultured from 18-days-old rats (Fig: 2.B). IBMX had no effect on lactate production by Sc at any

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age groups.

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Production of cAMP

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Amount of cAMP produced by 9-days-old Sc was poor at all time points when treated with GF

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media alone or GF containing FSH in absence of IBMX (Fig: 3. A). In presence of IBMX, FSH

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induced cAMP at 4hr was further elevated at 12hr and maintained upto that level at 24hr (Fig:

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3.B). On the other hand, in 18-days-old Sc, FSH induced cAMP production was observed even

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in absence of IBMX at all time points (Fig: 4.A). However, in presence of IBMX, cAMP was

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basally augmented at all time points in this age of rats. FSH significantly induced cAMP

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production at all time points and the levels remained constant from 2hr to 24hr (Fig: 4.B).

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Gene Expression profile

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Transcripts of transferrin were augmented by FSH in IBMX untreated group at 2hr, 4hr, 12hr in

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9-days (Fig: 5A) and at 2hr, 4hr, 8hr in 18-days-old rat Sc , (Fig: 6.A). However, in IBMX

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treated groups (for both the ages), the transcripts of this gene were basally elevated, probably

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because of excessive accumulation of cAMP and resulted into lack of further rise in the mRNA

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expression upon FSH treatment (Fig: 5.A and 6.A). At 24 hr., there seemed to be an inhibition.

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Inhibin β-B mRNA were augmented by FSH in IBMX untreated group at 2hr, 4hr in both 9-days

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(Fig: 5.B) and 18-days-old rat Sc, (Fig: 6.B). However, in IBMX treated group (for both age

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groups), the transcripts of this gene were also basally elevated (may be due to excessive

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accumulation of cAMP) resulted into lack of further augmentation in the mRNA expression upon

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FSH treatment (Fig: 5.B and 6. B). ABP mRNA were augmented by FSH in IBMX untreated 9-

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days-old rat Sc at 2hr, 4hr, 8h, and 12hr (Fig: 5.C) However, IBMX treatment in these cells

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elevated the basal transcription of ABP mRNA at all termination time points resulting into no

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FSH mediated augmentation of ABP mRNA (Fig: 5.C). In 18-days-old rat Sc, ABP expression

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was only augmented at 2hr of FSH exposure in absence of IBMX. In rest of the time points,

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basal levels of ABP mRNA were very high (Fig: 6.C). In presence of IBMX, FSH failed to

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augment ABP mRNA at all the time points in 18-days-old Sc (Fig: 6.C). Cyclophilin A was

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used as endogenous control as its expression remained unaltered with treatment or age of Sc3.

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Expectedly, its expression remained unchanged in both 9-days (Fig: 5. D) and 18-days-old Sc

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(Fig: 6. D) irrespective of IBMX treatment and termination points. Densitometric analysis of the

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expression of each gene, for each treatment, in each hr was determined in both the age groups,

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and collectively represented by the relative the expression of the target genes (transferrin, Inhibin

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β-B and ABP) against housekeeping gene cyclophilin A.

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Effect of hormonal supplementation on Sc culture-

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Morphology of cultured Sc in presence or absence of FT

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Morphology of Sc isolated and cultured from 9-days-old rats showed remarkable difference from

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that of Sc isolated and cultured from 18-days-old rats (Fig: 7.A and C). However, when 9-days-

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old rat Sc was cultured for 4 days in presence of FT (FSH and T in combination), there was a

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pronounced change in morphology and they resembled to 18-days-old rat Sc (Fig: 7.B, and D).

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Evaluation of the differentiation status of Sc in vitro with presence or absence of hormones (FT)

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MIS transcripts were detected on Day 0 of culture in 9-days-old rat Sc, however, the expression

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level decreased gradually when Sc were cultured with or without FT supplementations. (Fig:

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8.A). MIS mRNA were also detected on Day 0 of culture in 18-days-old rat Sc, however, the

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expression level were maintained even in absence of hormones in culture conditions. Continuous

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FT treatment for 4 days in vitro inhibited the expression of the transcripts (Fig: 8.A). The

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transcription of GDNF mRNA was decreased in culture of 9-days-old rat Sc compared to that of

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the freshly isolated Sc. FT supplementation in this culture resulted to a higher mRNA expression

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with a lower basal level as seen on day 0 (Fig: 8.B). However, a distinct augmentation of GDNF

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mRNA was detected after 4 days of FT supplementation in 9-days-old cultured Sc (Fig: 8.B).

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18-days-old Sc were able to express GDNF mRNA in culture even in absence of hormones. No

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change in expression of GDNF mRNA was observed in between freshly isolated Sc and cultured

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Sc in this age group (Fig: 8.B). Transcription of transferrin mRNA was dependent on hormones

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in 9-days -old rat Sc. The transcript levels were even higher in Sc cultured in presence of FT on

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day 0 than that of the freshly isolated Sc (Fig: 8. C). However, in 18-days-old rat Sc the

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transcript levels remained uniform in both freshly isolated Sc and cultured Sc (with or without

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FT supplementations) (Fig: 8. C). Both soluble (505 bp Sol) and membrane bound (420 bp

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Memn) isoforms (upper and lower bands respectively) of SCF mRNA were detected in 9-days-

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old Sc immediately after isolation. Continuous FT exposure in culture induced the expression of

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both the isoforms in this age group (Fig : 8.D ). Membrane bound isoform of SCF (the lower

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band) was dominant in 18-days-old Sc immediately after isolation. Although SCF mRNA was

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detected in FT untreated groups in 18-days-old rat Sc, FT mediated augmentation of SCF mRNA

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was discernible in these age groups in consecutive 3 days of culture (Fig: 8.D). Expression of

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cyclophilin A mRNA was evaluated as endogenous control and were found to be uniform in both

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direct assay and culture irrespective of the ages of rats (Fig : 8.E.).

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Discussion

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In the present study, we have investigated diverse parameters of Sc culture to understand

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hormone driven gene expression during the phase of postnatal maturation of Sc (from day 9

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onwards) essential for the onset of spermatogenesis. The first parameter was introduced by

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comparing the response of Sc (in terms of gene expression) immediately after the isolation from

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the testes (closer to in vivo) with the response of Sc which were cultured for 4 days (in vitro).

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The effect of hormones was also investigated by culturing Sc with or without hormonal (FSH

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and T in combination) supplements. Other parameters like different age groups (9-days-old

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neonatal and 18-days-old prepubertal rats representing immature and mature Sc respectively),

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different treatments (use of IBMX), different termination time points and the expression of

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various genes known to be either hormone (FSH and T) responsive or maturation markers of Sc

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were also included.

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Necessity of use of IBMX in Sc culture

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IBMX is a nonspecific inhibitor of phosphodiesterase (PDE)s broadly used in cell culture to

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obtain a better experimental readout9 . Although PDE4 specific inhibitor rolipram is available but

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IBMX has greater acceptance for use in Sc culture for decades9. Since, IBMX is not naturally

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present in the body it may appear to introduce artificiality to the experimental system. Apart

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from the accumulation of cAMP by inhibiting cellular PDEs, IBMX is also known to release

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Ca+2 from intracellular stores in neuronal cells10. Therefore, to investigate the appropriate

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hormonal (FSH) response in Sc, at first, it is essential to investigate the efficacy of using IBMX

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in Sc culture. We examined this logic while looking to determine various experimental end

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points such as secretion of metabolic products (lactate), production of cAMP by Sc and finally

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the expression of different FSH responsive genes. A possible nutritive role of Sc in Gc

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development is supported by the observations that Sc produce lactate at high rate and the rate of

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production increases further in presence of FSH11. Our result indicated that the lactate production

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by Sc was increased upon FSH treatment only in 18-days-old rats whereas FSH failed to

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augment lactate production in 9-days-old rat Sc. Such differential rates of lactate production

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observed at 9 days and 18 days of age also reflected the structural change in Sc12 with FSH

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responsiveness3 and altered oxidative stress13. However, IBMX had no effect upon the total

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accumulated lactate that was measured at 24hr of FSH exposure in both 9-days and 18-days-old

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rat Sc. This data suggested that lactate production by Sc might not be influenced by cAMP

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accumulation inside Sc. FSH is known to induce lactate production via PI 3-Kinase / PKB

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pathway in 20-days-old rat Sc14 and cAMP may directly activate PI 3-Kinase / PKB pathway

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without the involvement of cAMP induced PKA in Sc. IBMX directs accumulation of cAMP

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inside the cell by preventing its degradation via inhibiting PDEs. However, this excessive level

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of cAMP failed to augment lactate production by 9-days and 18-days-old rat Sc. So we

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concluded that when some biochemical metabolites (like lactate) is the experimental read out,

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addition of IBMX in Sc culture is unnecessary and has no benefit.

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FSH augmented cAMP production was observed in both 9-days and 18-days of age. However,

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unlike lactate production, the level of cAMP was further elevated by IBMX in FSH treated cells

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of 18-days-old Sc. Interestingly cAMP production by 9 days Sc is minimum and the level of

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cAMP produced by 9-days–old rat Sc upon FSH treatment was detectable only in presence of

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IBMX. Sc cultures from 10-days, 20-days, and 30-days-old rats have been treated with FSH in

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presence or absence of IBMX by Levallet et al15. FSH mediated cAMP production is reported to

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be highest in 20-days-old rats and a remarkable time dependent increase in the accumulation of

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cAMP is observed in presence of IBMX in this age group15. This report also suggests that

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endogenous activity of PDEs is highest at or around 20-days-old rats and the activity declines

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gradually with sexual maturity.

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Sc culture provides opportunity to investigate the expression of various genes under endocrine

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(FSH and T) or paracrine control8. In majority of studies, IBMX has been used in Sc cultures to

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detect gene expression upon treatments with FSH. Since T is not involved in cAMP generation in

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Sc [our unpublished observations] we treated the cells only with FSH in presence or absence of

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IBMX to determine the transcriptional augmentation of FSH responsive genes8 like transferrin,

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inhibin β-B and ABP (Androgen-binding protein or Sex hormone binding globulin SHBG).

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Transferrin is a transport glycoprotein that is essential for the delivery of iron to Gc within the

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adluminal compartment of the seminiferous tubules, Inhibin β-B is a subunit of Inhibin B, the

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major circulating inhibin in male rats, and ABP is a glycoprotein that specifically binds with

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androgen (T, in the testes)16. We found that FSH response in terms of the expression of

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transferrin, inhibin β-B mRNAs were more prominent in earlier time points (e.g. 2hr, 4hr ) in

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absence of IBMX in both the age groups. However ABP mRNA expression was not regulated by

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FSH in 18-days-old rat Sc. In IBMX treated groups, transcripts of these genes were basally

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elevated, probably due to constant accumulation of cAMP, resulting into lack of further elevation

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in the mRNA expression upon FSH treatment irrespective of ages of rats. Therefore, taken

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together, we concluded that IBMX should be used in Sc culture for measuring the cAMP

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response to FSH treatment by Sc, as the effective concentration of cAMP is determined both by

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its rate of synthesis and its rate of degradation effected by PDEs , whereas use of IBMX should

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be avoided for FSH induced gene expression analyses in Sc with an objective to extrapolate the

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outcome to in vivo situation.

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In vitro maturation of Sc by hormonal supplementations

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Although Sc culture is a well accepted tool for studying Sc functions for years, recently data

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from the Walker lab17,18 indicated a new system where Sc are used for experiments immediately

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after the isolation from the testes without culturing further. This new method is developed from a

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conviction that behavior of Sc changes in culture conditions due to the withdrawal of the factors

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(both endocrine and paracrine) present inside the seminiferous tubules19. However, by this

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technique, only the expression of genes in Sc at mRNA or protein level can be estimated3,17,18

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without further information like hormone mediated specific signaling pathways20-24. We

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therefore compared the changes in Sc behavior in terms of morphology and gene expression in

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freshly isolated Sc (closer to in vivo) and with traditional culture (in vitro) with or without

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hormonal (FSH and T in combination i.e. FT) supplementations for 4 days. The primary focus

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of this study was to investigate the influence of endogenous hormones (FT) to induce the

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expression of genes like MIS (Müllerian inhibiting substance), transferrin, GDNF (Glial cell

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line-derived neurotropic factor) and SCF (Stem Cell factor) as seen in freshly isolated Sc

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(influence of FT in vivo) and in cultured Sc with or without FT supplements ( i.e. influence of

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FT in vitro).

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MIS, also called anti-Müllerian hormone (AMH), a glycoprotein homodimer belonging to the

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transforming growth factor β superfamily, is a critical component of sex differentiation and

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responsible for the regression of the in the male embryo. Both MIS mRNA and protein remain

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high after birth and fall precipitously after day 5 to a low level, they remain throughout adult

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life25. We found that, MIS transcription was decreased progressively as Sc was cultured in vitro

307

for 4 days in both the age groups. Such decline in MIS expression in Sc culture was further

308

supported by previous observation by Arambepola et al.

309

continues FT supplementation for 4 days down regulated MIS mRNA expression more

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26

using 2-days-old rat Sc. However,

ACCEPTED MANUSCRIPT

prominently in 18-days of age as compared to that of the 9-days of age. This observation

311

suggested that 18-days old Sc are more sensitive towards hormones than 9-days old Sc. From

312

day 0 of isolation, the expression of MIS mRNA was higher in 18 days old Sc, although this may

313

not necessarily display a similar pattern in protein levels of MIS. GDNF, is a distantly related

314

member of the transforming growth factor-β superfamily, and contribute to the paracrine

315

regulation of spermatogonial self-renewal, differentiation and survival in the mouse27. The

316

transcription of GDNF mRNA was found to get decreased in culture of 9-days-old rat Sc only in

317

absence of hormonal supplements. However, FT treatment of the culture resulted into a

318

continued mRNA expression at a low level similar to that observed in freshly isolated cells in

319

this age. On the other hand, 18-days-old rat Sc was found to be capable to continue hormone

320

independent transcription of GDNF mRNA in vitro. This data also correlated with the age

321

dependent maturation of Sc in terms of an elevated expression of GDNF for the necessity of

322

enhanced self renewal and differentiation of the developing Gc in spermatogenically active

323

testis. Our observation of transferrin expression suggested its FT dependency in 9-days and

324

independency in 18-days-old rat Sc. However, it is important to note that the transcript levels

325

increased in Sc cultured for 4 days with continuous FT supplementation than that of the freshly

326

isolated cells from 9-days of age. The change in morphology in immature Sc with FT

327

supplementation also supported this gene expression data and provide the first demonstration of

328

FSH and T induced Sc maturation in vitro. Under the influence of FSH, Sc produce two

329

isoforms [soluble (sol) and membrane bound (Memn) upper (505 bp) and lower (420 bp) band

330

respectively] of SCF, which are indispensible for spermatogonial differentiation and survival28 .

331

Our results suggested that both of the isoforms of SCF were detected in Sc freshly isolated from

332

9-days-old rats and FT supplementation augmented their expression in culture. On the other hand

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only the membrane bound form is expressed in freshly isolated Sc from 18-days-old rats and the

334

soluble isoform appeared only after culture. This clear shift in the change in the alternative

335

splicing of SCF gene and the magnitude of FT induced augmentation of this gene with age,

336

indicated that an adequate gain in hormonal responsiveness occurs with postnatal maturation of

337

Sc.

338

Finally, in an attempt to reveal the hormone driven changes in Sc gene expression during

339

postnatal maturation, the present study for the first time have compared the efficacy of use of

340

freshly isolated and cultured Sc with and without hormonal supplementations. This work

341

provided substantial evidences of hormone derived in vitro maturation of rat Sc. Therefore, it is

342

recommended that for a comparative study of hormone mediated signaling in immature and

343

mature Sc, culture of immature Sc should ideally be performed in absence of hormones to retain

344

the developmental status of Sc intact or Sc should be used on day of isolation (day 0).

345

Alternately , one can culture 5 days old rat Sc and use them for 4 days in vitro ensuring that age

346

of Sc does not exceed 9-days (related to established immaturity of

347

maturation is taken into consideration. That would allow use of truly immature Sc for

348

comparison with other, mature age groups. Mature Sc from 18-days of age was found to be more

349

responsive towards FSH as compared to Sc from 9-days of age in terms of lactate, cAMP and

350

gene transcription. These maturational changes in Sc are necessary to promote the induction of

351

Gc differentiation at the time of onset of spermatogenesis. Additionally, this work also

352

highlighted about the need of restricted use of IBMX, specially in FSH induced gene expression

353

analysis in primary culture of Sc. It is also necessary to examine further whether IBMX (via

354

accumulation of cAMP) can induce in vitro differentiation of Sc.

Sc), even if in vitro

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ACKNOWLEDGMENTS

356

We are thankful to all the staff of the Small Animal Facility. Thanks are due to Ram Singh,

357

Dharamvir Singh, and Birendar Roy for technical assistance. We are grateful to Dr.

358

Bholashankar Pradhan for his assistance for cell imaging. We also thank Dr. A. F. Parlow

359

(NHPP, NIH) for providing the hormones used in this study. We are grateful to the Director of

360

NII for valuable support.

361

GRANTS

362

We thank the Department of Biotechnology and Indian Council of Medical Research,

363

Government of India, for funding.

364

DISCLOSURES

365

No conflicts of interest, financial or otherwise, are declared by the author(s).

366

AUTHOR CONTRIBUTIONS

367

IB and SSM designed the research, IB and MG performed the experiments, IB and SSM

368

analyzed the data and wrote the paper.

369

References

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2. Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 1994: pp. 1363 1434.

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3. Bhattacharya, I., Pradhan, B.S., Sarda, K., Gautam, M., Basu, S., Majumdar, S.S. A

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switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ

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cell differentiation during prepubartal testicular maturation in rats. Am. J. Physiol.

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Endocrinol. Metab. 2012; 303: E886-E898. 4. Majumdar, S.S., Sarda, K., Bhattacharya, I., Plant, T.M. Insufficient androgen and FSH

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signaling may be responsible for the azoospermia of the infantile primate testes despite

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exposure to an adult-like hormonal milieu. Hum. Reprod. 2012; 27: 2515-2525.

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5. Brinster RL and Zimmermann J. Spermatogenesis following male germ-cell transplantation. Proc. Nati. Acad. Sci. USA. 1994; 91 : 11298-11302.

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6. Gondos B and Berndston WE. Postnatal and pubertal development. In: Russell LD,

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7. Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of

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Sertoli cells, and their relevance to disorders of testis function in adulthood.

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8. Walker WH and Cheng J. FSH and testosterone signaling in Sertoli cells, Reproduction. 2005;130: 15-28.

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9. Mehats C, Andersen C.B, Filopanti M, Catherine Jin S-L. and Conti M . Cyclic

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nucleotide phosphodiesterases and their role in endocrine cell signaling. Trends in

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10. Usachev Y and Verkhratsky. IBMX induces calcium release from intracellular stores in

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11. Jutte N.H.P.M, Jansen R, Grootegoed J. A, Rommerts F. F. G and van der Molen H. J.

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FSH stimulation of the production of pyruvate and lactate by rat Sertoli cells may be

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involved in hormonal regulation of spermatogenesis. Journal of Reproduction and

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Fertility. 1983; 68, 219-226.

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12. Tung P.S, Dorrington J.H, and Fritz I.B. Structural changes inducted by folliclestimulating hormone or dibutyryl cyclic AMP on presumptive Sertoli cells in culture. PNAS. 1975; 72(5): 1838–1842.

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13. Raychoudhury S.S and Dana Kubinski D. Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response. Environ Health Perspect. 2003; 111(1): 33–38.

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14. Meroni SB, Riera MF, Pellizzari EH and Cigorraga SB. Regulation of rat Sertoli cell

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function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B

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15. Levallet G, Levallet J, Bouraima-Lelong H and Bonnamy PJ. Expression of the cAMP-

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hormone-stimulated PDE4 activities in immature rat Sertoli cells. Biol Reprod. 2007; 76

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:794–803.

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16. Bardin CW, Cheng CY, Mustow NA and Gunsalus GL. The Sertoli cell. In: Knobil E,

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Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 1994 .

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17. Wood MA and Walker WH. USF1/2 transcription factor DNA-binding activity is induced during rat Sertoli cell differentiation. Biol Reprod. 2009; 80:24-33.

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18. Upstream stimulatory factor induces Nr5a1 and Shbg gene expression during the onset of

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rat Sertoli cell differentiation. Wood MA, Mukherjee P, Toocheck CA, Walker WH. Biol

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19. Russell LD, Steinberger A. Sertoli cells in culture: views from the perspectives of an in vivoist and an in vitroist. Biol Reprod. 1989;41: 571–577.

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20. Crepieux P, Marion S, Martinat N, Fafeur V, Vern YL, Kerboeuf D, Guillou F and Reiter

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E. The ERK-dependent signalling is stage-specifically modulated by FSH, during

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primary Sertoli cell maturation. Oncogene. 2001; 20 : 4696–4709. 21. Fix C, Jordan C, Cano P and Walker WH. Testosterone activates mitogen-activated

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protein kinase and the cAMP response element binding protein transcription factor in

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Sertoli cells. Proc. Nati. Acad. Sci. USA. 2004; 101 :10919–10924.

22. Cheng J, Watkins SC, Walker WH. Testosterone activates mitogen activated protein

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kinase via Src kinase and the epidermal growth factor receptor in

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Endocrinology. 2007;148: 2066–2074.

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23. Viswanathan P, Michelle A. Wood and Walker WH. Follicle-Stimulating Hormone

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(FSH) Transiently Blocks FSH Receptor Transcription by Increasing Inhibitor of

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Deoxyribonucleic Acid Binding/Differentiation-2 and Decreasing Upstream Stimulatory

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Factor Expression in Rat Sertoli Cells. Endocrinology. 2009;150: 3783-3791.

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24. Regulation of Sertoli-germ cell adhesion and sperm release by FSH and nonclassical

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testosterone signaling. Shupe J, Cheng J, Puri P, Kostereva N, Walker WH. Mol

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Endocrinol. 2011;25:238-52.

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25. MacLaughlin DT and Donahoe PK. Sex determination and differentiation. N. Eng J. Med. 2004;22;350 (4):367-78.

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26. Arambepola N.K, Bunick D and Cooke P.S. Thyroid Hormone and Follicle-Stimulating

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Hormone Regulate Müllerian-Inhibiting Substance Messenger Ribonucleic Acid

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Expression in Cultured Neonatal Rat Sertoli Cells. Endocrinology. 1998; 139:4489-4495.

27. Hofmann MC. Gdnf signaling pathways within the mammalian spermatogonial stem cell niche. Mol Cell Endocrinol. 2008; 25; 288(1-2):95-103.

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446 447

28. Bedell M and Zama AM. Genetic Analysis of Kit Ligand Functions During Mouse Spermatogenesis. Journal of Andrology. 2004; 25:188-199.

Fig Legends

449

Fig1. Purity of Sc Culture. A. Phase of Sc culture obtained from 9-days-old rats. B. Nucleus

450

stained by hoechst in Sc culture obtained from 9-days-old rats. C. Vimentin staining in 9-days-

451

old Sc. D. 9-days-old Sc culture under red filter. E. Phase of Sc culture obtained from 18-days-

452

old rats. F. Nucleus stained by hoechst in Sc culture obtained from 18-days-old rats. G. Vimentin

453

staining in 18-days-old Sc. H. 18-days-old Sc culture under red filter. Each image of each age is

454

a representative of ten random snaps obtained from at least three independent sets of culture.

455

Fig2. Effect of IBMX on FSH induced lactate production by Sc cultured from 9-days and

456

18-days-old rats at 24hr.

457

A. Lactate production in 9-days-old Sc in presence or absence of IBMX B. Lactate production in

458

18-days-old Sc in presence or absence of IBMX. C = gf media only, F = gf media containing

459

50ng/ml o-FSH. C+ = gf media + IBMX (10-4M) only, F+ = gf media containing 50ng/ml o-FSH

460

+ IBMX (10-4M) only, (* = P < 0.05%).

461

Fig 3. Effect of IBMX on FSH induced cAMP production by Sc cultured from 9-days-old

462

rats at 2hr, 4hr, 8hr, 12hr, 24hr. A. cAMP production in 9-days-old Sc in absence of IBMX,

463

B. cAMP production in 9-days-old Sc in presence of IBMX, (*: P<0.5 %).

464

Fig 4. Effect of IBMX on FSH induced cAMP production by Sc cultured from 18-days-old

465

rats at 2hr, 4hr, 8hr, 12hr, 24hr. A. cAMP production in 18-days-old Sc in absence of IBMX,

466

B. cAMP production in 18-days-old Sc in presence of IBMX, (*: P<0.5 %).

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Fig 5. Effect of IBMX on FSH induced gene expression by Sc cultured from 9days-old rats

468

at 2hr, 4hr, 8hr, 12hr, 24hr. A. Expression of transferrin mRNA in absence and presence of

469

IBMX. B. Expression of inhibinβ-B mRNA in absence and presence of IBMX. C. Expression of

470

ABP mRNA in absence and presence of IBMX. D. Expression of cyclophilin A mRNA in

471

absence and presence of IBMX. Provided each gel picture (for each age group of rats) is a

472

representative of three sets of independent experiments. Provided bar diagrams are the relative

473

mRNA expression and were calculated by densitometric analyses of each target gene (transferrin

474

, inhibinβ-B and ABP) normalized against the endogenous control cyclophilin A for (* = P <

475

0.05%).

476

Fig 6. Effect of IBMX on FSH induced gene expression by Sc cultured 18days-old rats at

477

2hr, 4hr, 8hr, 12hr, 24hr. A. Expression of transferrin mRNA in absence and presence of

478

IBMX. B. Expression of inhibinβ-B mRNA in absence and presence of IBMX. C. Expression of

479

ABP mRNA in absence and presence of IBMX. D. Expression of cyclophilin A mRNA in

480

absence and presence of IBMX. Provided each gel picture (for each age group of rats) is a

481

representative of three sets of independent experiments. Provided bar diagrams are the relative

482

mRNA expression and were calculated by densitometric analyses of each target gene (transferrin

483

, inhibinβ-B and ABP) normalized against the endogenous control cyclophilin A

484

0.05%).

485

Fig:7. Phase Contrast Microscopy of Sc cultured from 9-days and 18-days-old rats for 4

486

days. A. Sc cultured from 9-days-old rats in absence of FSH and T. B. Sc cultured from 9-days-

487

old rats in presence of FSH and T. C. Sc cultured from 18-days-old rats in absence of FSH and T.

488

D. Sc cultured from 18-days-old rats in presence of FSH and T. All images are captured at 20x

489

magnification.

(* = P <

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Fig: 8. In vitro maturation of rat Sc. A. RT- PCR analyses of MIS mRNA expression in freshly

491

isolated and cultured Sc with and without FSH and T supplementation prepared from 9-days and

492

18-days of rats. B. RT- PCR analyses of GDNF mRNA expression in freshly isolated and

493

cultured Sc with and without FSH and T supplementation prepared from 9-days and 18-days of

494

rats. C. RT- PCR analyses of transferrin mRNA expression in freshly isolated and cultured Sc

495

with and without FSH and T supplementation prepared from 9-days and 18-days of rats. D. RT-

496

PCR analyses of SCF mRNA expression in freshly isolated and cultured Sc with and without

497

FSH and T supplementation prepared from 9-days and 18-days of rats, [two isoforms i.e. soluble

498

(sol) and membrane bound (Memn), upper (505 bp) and lower (420 bp) band respectively]. E.

499

RT- PCR analyses of cyclophilin A mRNA expression in freshly isolated and cultured Sc with

500

and without FSH and T supplementation prepared from 9-days and 18-days of rats. FI= Freshly

501

isolated Sc. -= without and + = with FSH and T (in combination) supplementation. Provided

502

each gel picture (for each age group of rats) is a representative of different sets of independent

503

experiments.

506

507

508

509

SC

M AN U

TE D EP

505

AC C

504

RI PT

490

ACCEPTED MANUSCRIPT

Table 2. List of Primer used

510

Acc. No.

SCF

Sequences (5’_3’)

NM_021844.1

Tm

GCT TGA CTG ATC TTC TGG ACA

AAC TGC CCT TGT AAG ACT TGG

GDNF

NM_019139.1

M AN U

C (R)

PCR

Size (bp)

Cycle

60°C 505 bp

420 bp

(Membrane bound)

ATGAAGTTATGGGATGTCGTGGCT 65°C 617 bp (F)

35

(Soluble)

SC

AG (F)

Product

RI PT

Gene

35

NM_012902.1

AGTTGCTAGTCCTACATCTGGC (F) 58°C 312 bp

35

EP

MIS

TE D

GGGTCAGATACATCCACACCG (R)

AGGCCTGCAGCTGAGCGATGGT

AC C

(R)

Transferrin NM_001013110.1 CCACATGAAAACCGTCCTTCC (F)

Inhibin ß-B NM_080771.1

66°C 401 bp

35

67°C 458 bp

35

AACTGCCCGAGAAGAAACTGG (R) AGCGCGTCTCTGAGATCATCA (F)

TCGGATGCGATGTCTGCTATC (R)

ACCEPTED MANUSCRIPT

ABP

NM_012650.1

ACAAGTTTCTGCATCCCTGGC (F)

67°C 510 bp

35

67°C 120bp

25*

TCCATCTTTGGTCCTTGGCTC (R) XM_341363.4

TCACCATTTCCGACTGTGGAC (F)

RI PT

Cyclophilin A

ACAGGACATTGCGAGCAGATG (R)

SC

511

* Lower cycle number for the house keeping gene reflects higher abundance of their

513

transcripts.

AC C

EP

TE D

M AN U

512

ACCEPTED MANUSCRIPT

Table: 1. Experimental Work Plan (for each age group) Days of Culture Day 1

Day 2

Day 3

Day 4

RI PT

Day 0

Termination of lactate, cAMP and gene expression at 24hr Cytochemical evaluation of purity of Sc culture

Sc without FT saved in Trizol for gene expression

Sc with FT saved in Trizol for gene expression

EP AC C

SC

Sc without FT s c with FT saved in saved in Trizol for Trizol for gene expression gene expression

Hypotonic shock to remove Gc

In vitro treatments for FSH induced lactate, cAMP and gene expression in presence or absence of IBMX for 2-24hr

Sc without Sc morphology FT saved in with or without FT Trizol for gene expression

M AN U

Culture continued in 1% GF

TE D

Castration, Sc isolation and culture in 1% FCS with or without FSH and T (FT) FI= freshly Sc with FT isolated Sc saved in saved in Trizol Trizol for for gene gene expression expression

ACCEPTED MANUSCRIPT Table: 1. Experimental Work Plan (for each age group)

Days of Culture Day 2

Day 3

RI PT

Day 1

Day 4

M AN U

SC

Day 0

TE D

Cytochemical evaluation of purity of Sc culture

EP

Culture continued in 1% GF

AC C

Castration, Sc isolation and culture in 1% FCS with or without FSH and T (FT) FI= freshly Sc with FT isolated Sc saved in saved in Trizol Trizol for for gene gene expression expression

Termination of lactate, cAMP and gene expression at 24hr

sc with FT Sc without FT saved in saved in Trizol for Trizol for gene expression gene expression

Hypotonic shock to remove Gc Sc without FT saved in Trizol for gene expression

Sc with FT saved in Trizol for gene expression

In vitro treatments for FSH induced lactate, cAMP and gene expression in presence or absence of IBMX for 2-24hr Sc without Sc morphology FT saved in with or without FT Trizol for gene expression

B

C

D

SC

A

RI PT

ACCEPTED MANUSCRIPT

G

H

EP

F

AC C

E

TE D

M AN U

9-days-old immature Sc

18days-old mature Sc

Fig: 1. Vimentin staining and purity of Sc culture

ACCEPTED MANUSCRIPT

RI PT

A

M AN U

SC

9days-old Immature Sc

C-

F-

F+

TE D

B

C+

AC C

EP

18days-old mature Sc

C-

F-

C+

F+

Fig: 2. Effect of IBMX on FSH induced Lactate production

ACCEPTED MANUSCRIPT -IBMX

c A MP fmole/ml/million 9d S c IB MX

A 3 2.5

RI PT

2 1.5

SC

1

0 C 2hr

F 2hr

C 4hr

F 4hr

M AN U

0.5

C 8 hr

F 8hr

C 12hr

F 12 hr

C 24 hr

F 24 hr

TE D

3

EP

2.5 2

AC C

B

c A MP fmole/ml/million 9d S c + IB MX

+IBMX

1.5 1 0.5 0 C 2hr

F 2hr

C 4hr

F 4hr

C 8 hr

F 8hr

C 12hr

F 12 hr

C 24 hr

F 24 hr

Fig3. Effect of IBMX on FSH mediated cAMP production by 9-day s-old immature Sc

ACCEPTED MANUSCRIPT -IBMX

A

2.5

RI PT

2 1.5

SC

1 0.5 0 C 2hr

C 4hr

F 4hr

C 8 hr

F 8hr

C 12hr

F 12 hr

C 24 hr

F 24 hr

F 8hr

C 12hr

F12 hr

C 24 hr

F24 hr

+IBMX

B

TE D

10

F 2hr

9 8

EP

7 6 5

AC C

c A MP fm o le/m l/m illio n 18d S c + IB MX

M AN U

c A MP fm o le/m l/m ill io n 18d S c -IB MX

3

4 3 2 1 0 C 2hr

F 2hr

C 4hr

F 4hr

C 8 hr

Fig4. Effect of IBMX on FSH mediated cAMP production by 18-day s-old mature Sc

A

ACCEPTED MANUSCRIPT

9days-old immature Sc Transferrin

-IBMX

**

*** ***

Transferrin

** *

- IBMX

Inhibin βB

**

M AN U

-IBMX *** ***

+IBMX ABP

TE D

C

AC C

+IBMX

-IBMX

Inhibin βB

Cyclophilin A

**

- IBMX

EP

-IBMX

D

+IBMX

SC

B

RI PT

+IBMX

+IBMX

*** ***

**

ABP

*

**

*

+IBMX - IBMX

+IBMX

2hr 4hr 8hr 12hr 24hr Fig 5. The effect of IBMX on FSH induced gene expression at 2hr to 24hr

*** ***

-IBMX

Transferrin **

*** *** **

+IBMX

- IBMX

Inhibin βB

SC

B

RI PT

A

ACCEPTED MANUSCRIPT

18days-oldmature Sc Transferrin

M AN U

-IBMX

+IBMX

*

***

+IBMX

TE D

ABP

-IBMX +IBMX Cyclophilin A

- IBMX

+IBMX

**

ABP

AC C

D

EP

C

Inhibin βB

-IBMX +IBMX C

F C F C F C F C F

- IBMX

+IBMX

2hr 4hr 8hr 12hr 24hr Fig 6. The effect of IBMX on FSH induced gene expression at 2hr to 24hr

ACCEPTED MANUSCRIPT

- (FSH+T)

+(FSH+T) B 9days-old immature Sc

M AN U

SC

RI PT

A

C

18days-old mature Sc

AC C

EP

TE D

D

Fig:7. Morphological change of Sc upon hormonal supplementation

Days of culture A

1ACCEPTED MANUSCRIPT 2

0 FI

-

+

-

3

+

-

+

9day s-old immature Sc

MIS

RI PT

18days-old mature Sc B

SC

9day s-old immature Sc

18days-old mature Sc E 9day s-old immature Sc

EP

9day s-old immature Sc

AC C

D

TE D

C 9day s-old immature Sc 18days-old mature Sc

GDNF

M AN U

18days-old mature Sc

Transferrin Sol SCF Memn SCF

Cyclophilin A

18days-old mature Sc Fig: 8. In vitro maturation of Sc upon hormonal supplementation