Role of proteins secreted from F tubes in capitation of sperms : What we need to know?

 

Poster presentation: Copied    from Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010

  P-007: Can some beneficial proteins help in capacitation of sperms to make the sperm more fertilized capacity. But how then we explain in IVF medium where such tubal secretions which help in capacitation of sperms over hours are absent ??  Isolation and identification of a protein from human  tubal (oviductal )  secretion that interact with human spermatozoa

What was the conclusion?? Conclusion: At least five oviductal proteins that interact with sperm membrane

were detected (127 kDa, 94 kDa, 79 kDa, 17 kDa and 14 kDa). In the protein

extract from sperm incubated in the presence of CM a protein of approximately

14 kDa was also present. This protein was identified as human calgranulin B

and it was detected both in CM and nOF by Western blot. These results support

the hypothesis that some oviductal proteins could modulate the sperm function

through direct binding on sperm and could have an active role in the fertilization

process.

 

 

C. Zumoffen1, M.J. Munuce1, A. Caille1, S. Ghersevich1

 

1   School of Biochemical and Pharmaceutical Sciences, Laboratory of Reproductive

Studies - Clinical Biochemistry, Rosario, Argentina

 

Introduction: After ejaculation, a number of spermatozoa move to the first

portion of the oviduct, where they can remain viable during hours or even

days in contact with the oviductal secretion and can undergo several metabolic

and functional changes to become fertilizing-competent (process known as

capacitation). We have found that proteins from conditioned medium (CM) of

human oviductal tissue cultures can modulate some sperm functions. It have

been suggested that some oviductal proteins interact with human spermatozoa

and could modulate sperm function. Thus, the aim of the present study was to

isolate and identify proteins from CM with capacity to interact with human

spermatozoa.

 

Materials and Methods: Human oviductal tissue was obtained from pre-

menopausal

women (age: 41.0 ± 1.6, n =   20) with no clinical history of infection

or cancer disease, scheduled for routine hysterectomies. Native human oviductal

fluid (nOF) was recovered by flushing the tubes with DMEM/Ham F12 medium.

Explants of tubal tissues were cultured in DMEM/Ham F12 medium at 37 ºC

and 5 % CO2 for 24 h, followed by a further incubation with [35S]Met (30 µCi/

ml) during 24 hs, to obtain de novo [35S]Met-proteins. After incubation, CM was

collected and centrifuged 5 min at 700 × g, to remove debris. The CM were then

dialysed, lyophilised and stored at -70ºC until use. Total protein concentration in

CM was determined using the Bradford´s assay. Human spermatozoa were obtained

from normozoospermic samples of healthy donors (n = 4) after 3 days of

sexual abstinence. Motile sperm were recovered by swim-up and were incubated

under capacitating conditions in Ham F10 medium supplemented with 3.5 %

BSA for 2 h in the absence or the presence of [35S]Met-proteins from CM. At the

end of incubations, sperm membrane proteins (SMP) were extracted, analysed

by SDS-PAGE (10%) and electrophoretically transferred to nitrocellulose membranes.

In addition, a chromatographic affinity column was prepared with motile

sperm membrane extracts coupled to Sepharosa 4B, in which CM was seeded.

The eluted protein fractions were subjected to SDS-PAGE 10%. The protein

bands were analysed by LC-MS/MS. The presence of the identified protein in

nOF and CM was examined by Western blot analysis.

 

Results: A protein band with an estimated molecular weight (MW) of 14 kDa

was detected in the autoradiographies of SMP from spermatozoa that were incubated

with [35S]Met-labelled oviductal proteins. The SDS-PAGE of eluted

protein fractions obtained by affinity chromatography showed the presence of

at least five [35S]Met-protein bands by autoradiography, with estimated MW of

127 kDa, 94 kDa, 79 kDa, 17 kDa and 14 kDa, respectively. The 14 kDa protein

was identified as human calgranulin B by LC-MS/MS analysis. The presence

of calgranulin B in CM and nOF was confirmed by Western blot using specific

antibodies.

 

Conclusion: At least five oviductal proteins that interact with sperm membrane

were detected (127 kDa, 94 kDa, 79 kDa, 17 kDa and 14 kDa). In the protein

extract from sperm incubated in the presence of CM a protein of approximately

14 kDa was also present. This protein was identified as human calgranulin B

and it was detected both in CM and nOF by Western blot. These results support

the hypothesis that some oviductal proteins could modulate the sperm function

through direct binding on sperm and could have an active role in the fertilization

process.