Principal Investigator :    Subeer S Majumdar

Project Assistants
Minu Sinha

PhD Students
Y Sangeeta Devi
Kanchan Sarda

Collaborators
Tony M Plant, Univ Pittsburgh, USA

The theme of this project is to understand mechanism of Sertoli cell (Sc) regulation of spermatogenesis in primates and rodents, and paracrine interaction within the testis. This information would lead to proper evaluation and management of human infertility caused due to seminiferous tubular dysgenesis which is untreatable at present. Additionally, several unknown factors of seminiferous tubular origin important for the occurrence of spermatogenesis in mature testis may be identified by this study and used as a tool for achieving non-hormonal contraception in the males. The objectives of this study are to (i) evaluate functions of Sc from spermatogenetically inactive (infant) and active (pseudo-adult) monkey testis, (ii) compare Sc functions from these two developmental stages to identify processes and factors important for spermatogenesis, (iii) study testicular cell-cell interaction in vitro and (iv) undertake similar studies using 10 & 40 days old rats.

A remote blood sampling unit is established at PRC. This system is unique of its kind and established for the first time in this country. An intravenous indwelling catheter is surgically inserted under aseptic condition and implanted permanently for a fixed period. Catheter is exteriorized at the back and transferred to the adjacent room through a minute hole (in the wall) and patency of the catheter is maintained using a jacket, cable and swivel.

Such catheter can be used for injection of hormone or drugs and they can also be utilized for frequent blood sampling. RBCs can be returned back to monkey after collection of plasma. Several endocrinological and pharmacokinetic studies can be rationally undertaken using such system. Four pseudo-adult monkeys were produced by infusion of pulsatile (one pulse every three hr) GnRH for four weeks to juvenile male rhesus monkeys at NII. The testosterone levels were in the range of adult monkeys by the end of 4 weeks of treatment. Additionally, interstitial cells obtained from such monkeys, by collagenase digestion, were found to be highly responsive to exogenous hCG and like adult, produced significantly higher levels (4-5 fold) of T due to hCG treatment. This proves successful functioning of this unit at NII.

In our previous report we have shown that recombinant human (rh) FSH failed to stimulate production of lactate by juvenile bonnet monkeys in our hands. Considering that those Sc cells failed to respond to FSH because of a) Juvenile nature of Sc b) heterologous hormone (rh FSH), we treated adult like Sc isolated from GnRH primed (4weeks) rhesus monkeys with recombinant monkey (rm) FSH. Surprisingly, these pseudoadult Sc failed to augment lactate production upon addition of increasing concentration of rmFSH (5,10 and 20 ng /ml). It is important to note that circulating levels of FSH in adult rhesus monkeys is 2-5 ng/ml. Sertoli cells from two infant monkeys were also cultured. LNCaP and DU145 (or CV1) cell lines, which are positive and negative controls for androgen receptor, are being cultured in the laboratory for carrying further studies relevant to androgen receptor activity.

In vitro synthesized proteins were metabolically labeled with 35S methionine to compare secretory pattern of the pseudoadult and infant Sc. Equal amounts of radiolabelled proteins (50,000 cpm) obtained from culture supernatants were analyzed by 2D-PAGE followed by fluorography. pI and molecular weight were deduced from reference 2D markers run parallel with the samples.

Hormonally regulated secreted proteins from infant and pseudoadult monkeys were analyzed. In both the groups of monkeys, no new protein (s) were secreted when the cells were cultured in the presence of hormones (FSH+ T) as compared to cells cultured without hormones. However, relative intensity of some of the proteins changed upon hormone treatment. When hormone treated Sc proteins from infant and pseudo-adult monkeys were compared, intensity of approximately 70kD protein (pI-4) was found lower while a 45kD protein (pI-4) and a 50kD protein (pI 5.3) were expressed in higher intensities, in infants. A group of proteins at approximate pI of 5.5 and molecular weight of 66kD and another protein at approximate pI of 5.8 and molecular weight of 38kD were present specifically in neonates but not in the adult Sc media. Similarly, a group of proteins with approximate molecular weight of 40kD and pI of 4 -5 was expressed only in pseudo-adult monkeys.

Publications

Original peer-reviewed articles

1.     Nijhara R, Jana SS, Goswami SK, Rana A, Majumdar SS, Kumar V and Sarkar DP (2001) Sustained activation of mitogen-activated protein kinases and activator protein 1 by the hepatitis B virus X protein in mouse hepatocytes in vivo. J Virol 75:10348-10358.