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Functional comparison of Sertoli cells isolated from spermatogenetically active and inactive testis to elucidate role of Sertoli cells in spermatogenesis and infertility |
| Principal Investigator : Subeer S Majumdar
Project Associates
/ Assistants PhD
students Collaborators 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 the project are to (i) evaluate functions of Sc from spermatogenetically inactive (infant) and active (pseudoadult) monkey testis, (ii) compare Sc functions from these two developmental stages for identifying processes and factors important for spermatogenesis, (iii) study testicular cell-cell interaction in vitro and (iv) undertake similar studies using 10 & 40/60 days old rats. Hormonal Regulation FSH Lack of FSH responsiveness of monkey Sc was confirmed further when no rise in lactate production was discerned by addition of either (1) 5-fold glucose in the culture media or (2) 8-bromo cyclic AMP. Secretion of Sc inhibin B, which is known to be regulated by FSH, was also not augmented by rmFSH treatment in vitro. RNase protection assay (RPA) was used to analyse Sertoli cell mRNA from rmFSH treated and untreated cells. FSH failed to augment the expression of Inhibin bB mRNA, which corroborates with the findings of inhibin B secretion from these cells in response to FSH. Androgen Androgen receptor (AR) binding studies have indicated that Sc from infant monkeys are relatively incompetent to bind radioactive androgen as compared to that from pseuodoadults. To measure AR activity, we used recombinant adenovirus for delivering an androgen-responsive reporter gene (MMTV-luciferase). Luciferase expression by Sc transfected with adenovirus had problems because androgen treatment failed to induce luciferase expression. However, infection with AR-his (which express AR in the cells) followed by MMTV-Luciferase resulted into significantly higher binding and luciferase expression in pseuodoadult (n=1) Sc. Immuno-cytochemistry Immuno-histochemical localization studies are being undertaken. Until now, we have used Sc from one animal of each group to evaluate relative expression of AR, FSH receptor, phospho-CREB (cyclic AMP response element binding protein) and SRC-1 (steroid receptor co-activator). Although nuclei of Sc had some background staining with pre-immune serum, cytoplasm of infant as well as pseudoadult Sc had distinct staining for FSH receptors indicating its presence in both groups of monkeys. CREB-p (an important molecule of FSH signal transductional pathway) expression was also equally detected, in the nucleus of, both the groups. AR receptors [detected using AR (c-19) polyclonal antibody against carboxy terminus of human androgen receptor, from Santacruz] and coactivator Src-1 were localized in nucleus as well as cytoplasm in infant. Their absence in Sc cytoplasm of adults indicates their recruitment to nucleus, which may be related to active status of mature Sc. However, we need to evaluate Sc from more animals to substantiate our observation. Additional cultures in 8-chambered slides are being done for this purpose. From the available data we hypothesize that the AR are poorly expressed and less active in the infant monkey Sc thereby affecting their capability to support germ cell proliferation and differentiation. Protein Analysis Infant
and pseudoadult Sertoli cells expressed different pattern of secretory
proteins. During this year, we have begun to use cellular proteins also in
addition to secretory proteins for 2D PAGE analysis. Confirmatory studies by
repeated 2-D PAGE of Sc secreted proteins has revealed that SGP-1, SGP-2 and
transferrin-like proteins were expressed by both developmental groups.
Intensities of SGP-1 and transferrin-like proteins were enhanced in
pseudoadult monkeys as compared to that in infants. Four proteins, yet to be
identified, having pIs ranging from 5.1 to 6 and approximate mol. wt. of 25kDa
to 45kDa were more abundant in infants. Two groups of proteins, assigned as
ScS1 and ScS2, which have a similar mol. wt. (~55kDa) and pI of 5.4 and 7.0
respectively, were observed only in infants while ScS3 and ScS4, proteins with
pIs of 5.3 and 5.7 and mol. wt of 25kDa (both) were present only in the
pseudoadult monkeys. Similarly, infant (10 days old) and adult (60 days old) rat Sc secretory proteins were compared. SGP-1, SGP-2 and transferrin were found increased with age while some other unidentified proteins were decreased. In infants, a major portion of the total protein was occupied by a group of protein at the pI range of 4 - 5 and approximate mol. wt. of 45 kDa, which was comparatively less abundant in adults. Proteins specifically present in one developmental group were also observed. Two groups of proteins at pI range of 5.1 (mol. wt. 40kDa) and 6.0 (mol. wt. 45kDa) were present only in infants while protein at pI 5.1 and mol. wt. of 24 kDa was observed only in adults. We are in the process of sending the stage specific proteins for MALDI-TOF analysis by a company specialized in this field. Cell-cell interaction in the testis Study of cell-cell interaction in monkey Sertoli cells was difficult to undertake as very few cells could be isolated from limited number of testis. However, we have standardized Leydig cell and Peritubular cell cultures from rat testis. |