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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 |
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Principal Investigator : Subeer S Majumdar Project Associate Ph D students The aim of this project is to understand the
mechanism of the Sertoli cell (Sc) regulation of spermatogenesis in primates
and rodents. This study would help 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 occurrence of spermatogenesis in mature testes may be
identified and used as a tool for achieving non-hormonal contraception in the
males. Leydig isolation from rat and monkey Leydig cells from rats and monkeys were isolated
as a concentrated population using ficoll-paque. Although the fraction was
rich in 3-bHSD staining (specific for Lc) the T
production by this fraction was not very high as compared to that in crude
mixture of cells. However, use of percoll gradient (instead of ficoll) helped
in getting pure population of Lc from adult rats (60 days old) which produced
high amounts of T in vitro. Three bands appeared on percoll gradient of
which 2nd band (fraction II)
contained 80% or more Lc and T production of this fraction was 3-4 fold of
crude fraction loaded for such separation on percoll gradient. A dose response
study of hCG treatment confirmed their functional integrity after this
separation. Highest degree of stimulation was achieved with 400 pg hCG
treatment to approximately 1 million cells. However, occasionally this percoll
fraction showed reduced T production. To rule out harmful effect of percoll,
crude Lc were exposed to percoll and collected after dilution and pelleting.
These cells showed normal T production, which was comparable to unexposed
crude Lc, hence indicating that percoll is not responsible for occasional loss
of Lc activity after such isolation. We are beginning to isolate Lc from monkeys
using this procedure and also trying to detect cause of occasional Lc
disability to produce T in fraction II. Media from rat Lc cultures of fraction
II, showing good response to hCG, were added to Sc culture for evaluating role
of Lc secreted factors on Sc function. However, no encouraging change in
lactate production by Sc or aromatizing ability of Sc were noticed due to such
treatment. Similar studies in monkeys are being undertaken. Secreted proteins of Sc origin We have shown earlier that Sc of rat and monkey
from various age groups maintain functional integrity in vitro. We have
also shown in the past that production of few secreted proteins (as analyzed
by 2D PAGE) from 18 days old rats were augmented due to hormone treatment.
However, we had difficulties in getting enough amounts of secreted proteins
for proper analysis. To overcome this, now we collect the media from
radioactive methionine treated Sc for three consecutive days (days 5, 6 and 7
of cell culture) and pool them for concentrating on Centri-prep concentrator
(10 kDa cut off ). This has resulted into better yield of proteins and now we
have comparative data from 10 days, 40 days, and 60 days old rats. The age
related differences in secreted proteins were noticed. We plan to focus our
attention to these proteins, specific to one age group, and characterize them.
Pseudo adult monkeys Adult Sc culture from monkeys was done
successfully but the yield was not enough to undertake studies of protein
analysis. Therefore juvenile (12-20 months old) rhesus monkeys, in which the
hypothalamus is quiescent, were treated with GnRH pulses (0.3 mg/three
hours) for 4 weeks after surgical implantation of intravenous indwelling
catheters. This resulted into adult like hormonal pattern in infants and such
testis is known to show sign of germ cells differentiation. Since such testis
has adult like differentiated germ cells but minimal or no interfering
population of spermatocytes, spermatids and sperms, we cultured mature Sc from
such pseudo-adult testis with ease. We are first to do Sc culture from such
hormonally stimulated testis. To our benefit these cells attach well and
multiply vigorously. This gave us large number of culturable adult like Sc and
opened avenues to understand Sc function of adult, which is barely studied in
the past due to difficulties in its isolation and culture. Signal transduction in mature and immature Sc Infant rhesus monkey Sc was cultured from 2 and
4-month-old monkeys. Definitive functional studies are initiated in monkeys.
The measurement of cAMP level (using antibody from NIH in a radio-immunoassay)
after recombinant monkey FSH treatment to infant and pseudo adult Sc revealed
that highly active FSH receptor is expressed by pseudo-adult Sc as compared to
negligible by infant Sc. Similarly, androgen receptor studies indicated that
radioactive androgen could bind more efficiently to pseudo-adult Sc as
compared to infant. This lack of FSH responsiveness and androgen binding in
infants may be an important cause of spermatogenetically inactive testis in
infants in the phase of high levels of hormones. |