RBMY is expressed in developing germ cells and mature sperm cells. Immunofluorescence staining showed expression of RBMY in spermatogonia, spermatocytes, and elongating spermatids. In mature sperm, RBMY was detected in the midpiece and tail region. Incubation of sperm with RBMY antibodies did not affect motility parameters, indicating RBMY is not directly involved in sperm motility. RBMY plays a role in nuclear RNA processing during germ cell development based on structural domains. Deletion of the gene cluster containing RBMY leads to failed spermatogenesis and infertility in human males.

1. CELLULAR ONTGENCY OF rbmy during human spermatogenesis and its role in sperm
mobility.
Testicular tissue was minced and initially digested in collagenaseiv(1 mg,mL, Sigma USA,
St.Louis, USA) and subsequently in 0.25% Trypsin/EDTA at 370C. The cells were filtered
through 100,70 and 40 mm nylon filters and the flow through containing the germ cells was
washed thrice in PBS and used for immunofluorescence.
INDIRECT IMMUNOFLUORESCENCE
Indirect immune fluorescence:-
The protocol for immune fluorescence has been detailed preciously washed spermatozoa or
isolated germ cells were smeared, air-dried and fixed in child acetone. The slides were incubated
in 5% bovine serum albumin (BSA) and the cells were probed ovemight at 40c overnight with a
goat polyclonal antibody that recognizes the N-terminus of human RBMY (N-RBMY) at a
dilution of1:50. For negative control, normal goat serum was used inplace of the primary
antibody.
INDI
The different germ cell types were indetified under DIC based on morphology.
The Large cells with high nucleo- cytoplasmic ratio and fine chromatin were con – sidered
spermatogonia
Meiotic cells were identified based on their size and presence of chromo – serves. Round
spermatids were small interphase cells with condensed nucleus. Elongating spermatids were
indentified based on shape, placement of cytoplasm, condensed nucleus and the bail.
Double immunaluorescence:
To differentiate between spermatogania and spermatocytes me cells were immunostainal as
above for RBMY along with CKRT.
For detection of CKIT, a monoclonal antibody against CKIT(CELL Signalling, Danvers, USA)
was mixed along with the RBMY antibody.

2. Post washing. The slides were incubated with Frrc – labeled antigoat Iger for RBMY and Alexa
fluor 594 - labelled amto – mouse iger to detect Ckrr.
Post stringency washes in PBS – T the slides were counterstained with DAPI, mounted and ob –
served as above.
The negative controls included Slides incubated with the secnday antibodies alone.
2.7 Spumatogenesis:
Genetic analysis of men with infertitity and subfertility has to identification of genes that are
crucial fr spermato – gebesis.
The Y – Chromosome – encoded gene RBMY [RNA - binding motif on y] which is present in
multiple copies and distributed throughout they – chromosome,
The sequence and cellular distribution of RBMY protein are consistent with their function in
nuclear RNA processing. The open reading frame of RBMY CDNA contains an RNA – binding
domain and the carboxy – terminal domain has four repetitions of a ser – arg – croy -
Tyr tetrapeptice motif called SRGYa box which is a characteristic of many RNA – binding
proteins.
The murine and the human RBMY – RNA interactome has been recently reported Zeng etc
Structure of RBMY Complexed with RNA has also been solved.
Role of sperm motility
Functional studies have revealed that RBMY may play a role in sperm motility.
4.MATERIALS AND METHOD:
Sample collection and processing

3. Human testicular tissues were collected after informed consent and were a part of previous
studies.
Seman samples wer collected by masturbation from a anonymous dners. And those samples
meeting the criteria of normozoospermia were included in the study
All experiements were repeated twice using three independent samples.
Result
Expersion OF RBMY in developing testicular:-
In cells with the elongating tail the expression was found.
In the terminal stages of spermiogenesis where the cytoplasm was found to the extracting RBMY
was present in the cytoplasmic droplet.
The corresponding negative controls did not show any green fluorescene indicating the
specificity of the staining.
Using both the antibodies, intense fluorescene for RBMY was detected in the mid-piece of all
spermatozoa with weak but specific staining in the tail regin
No signals were detected in the negative controls.
EFFECT OF RBMY anti bodies on sperm motility
To determine any involvement of RBMY in sperm motility washed spermatozoa was incubated
with either normal rabbit/goat sesum or sesa containing the N-RBMY or the 1.RBMY
antibodies and the motility was determined by CA.SA.
Since the motility parameters of the cells incubated with rabbit or goat serum at the specified
dilutions did not differ significantly the data of both these control groups were pooled and
designated as control.
The indices did not differ significantly in case of cells incubated into 1RBMY antibody as
compared to controls.

4. Discussions
The results of the present study demonstrate the RBMY is expressed in all the germ cell types of
the testis and also in the ejaculated spermatozoa.
The y-chromosome has been thought to have evolved from an accident autosomal homoleguos
chromosome and in the course has lost most of its genes except these involved in male germ cell
developments.
Human males with deletion of the AZFb locus that harbours the RMBy genes have failure of
spermatogenesis are infertile.
INTRODUCTION
Spermatogenesis analysis f men with infertility and subfertility has led to identification of genes
that are crucial for spermato genesis. Is the y chromosome encoded gene RBMy C RNA-binding
mtif on y ) which is present in multiple copies and distributed throughout the y chromosome.
The sequence and cellular distribution of RBMy protein are consistent with their function in
nuclear RNA processing.
The open reading frame of RBMy CDNA contains an RNA binding domain and the carboxy
terminal domain has four repetitions of a ser-arj-gley, Tyr tetrapepetide motif called SRGy boox
which is a characteristic of many RNA-binding proteins.
The murine and the human RBMY-RNA interactome has been recently reported (Zeng et
al.2008-2011) the NMR structure of RBMy complexed with RNA has also been solved.
In the testicular germ cells . the process of active transcription and transaction occurs in the
premeicte and meiotic stages. The germ cells are transcriptionally and translationally quiescent
post meiosis
MATERIAL AND METHODS
SAMPLE COLLECTION ADNPROCESSING

5. Human tesitculat tissues were collected after informed consent and wer a part of previous
studies. Semem samples were collected by masturbation from anonym ous cloners, and those
samples meeting the criteria of normaozspermia were included in the study. All experiements
were repeated twice using three independent sample.
CELLULAR LOCALIZATION OF RBMy
Germ cell isolatin :- testicular tissue was minced and initially digested in collagenase iv and
subsequently in 0.25% trypsin /EDTA at 370c. the cells were filtered through 100,70 and 40 m.
nylon filters and the flow through containing the germ cells was washed thrice in PBS and used
for immunofluorescence.
Double immunofluorosence:
To differentiate between spermatogonics and spermatocytes. The cells were immunostated as
abve for RBMY along with CKIT.
Pst stringency washes is PBS-T. the slides were cuntersteuned with DAP1. Mounted and
observed as above. The negative controls intruded slides incubated with the secondary antibodies
alone.
Expression of RBMY in the developing tesficular.
In cells with the elongating tail expression was found. In the terminal stages of spermiogenesis
where the cytoplasm was found to the extracting RBMY was present in the cytoplasmic droplet
The corresponding negative controls did not show any green fluorescence indicating the
specificity of the staining .
Using both the antibodies intense fluorescence for RBMY was detected in midpoint of all
spermatoma with weak but specific staining in the tail region signals were detected in the
negative controls.
Effect of RBMY antibodies on sperm molitity.

6. To determine any involvement of RBMY in sperm motility washed spermatozoa were incubated
with either normal rabit/goat serum or sera containing the N-RBMY or the 1-RBMY antibodies
and the motility was determined by CA-SA.
Since the motility parameters of the cells incubated with rabbit or goat serum at the specified
dilutions did not differ significantly the data of bot. these control groups were pooled and
designated as control.
The indices did not differ significantly in case of cells incubated with 1 RBMY antibody as
compared to controls.
4. discussion:
The result of the present study demonstrate the RBMY is expressed in all the germ cell tyes of
the testis and also in the ejculated spermatozoa.
The y- chromosome has been thought to have evolved from an ancient autosomal homologous
chromosome and in the course has lost most of its gens except these involved in male germ cell
developments .
Human males with deletions of the AZFB locus that harbouls the RMBY genes have faiture of
spermatrgenesis are infertile.