Apical Region Of Sperm Head Research Articles

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing

The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species.

Role of TRPV1 channels in boar spermatozoa acquisition of fertilizing ability

Recently the transient receptor potential vanilloid type 1 (TRPV1) has been described to be involved in the capacitation, the process leading mammalian spermatozoa to acquire full fertilizing ability within the female genital tract. TRPV1 immunolocalization during capacitation and the effect of TRPV1 inhibition by the capsazepin (CPZ) or activation by the capsaicin (CPS) on membrane resting potential, calcium clearance and actin polymerization have been investigated. It was found that the capacitation promoted the translocation of TRPV1 from the post-acrosomal to the apical region of sperm head. Moreover the CPZ induced the progressive drop in intracellular Ca2+ levels during capacitation and the inhibition of actin polymerization in the acrosomal region. On the contrary, the CPS caused the sperm membrane depolarization due to the Na+ influx and the consequent voltage gated calcium channels (VGCC) opening. In conclusion it was suggested that TRPV1 channels modulate the major pathways involved in capacitation.

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes.

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.

Members of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are present and active in human sperm

Objective: To investigate whether components of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are present and active in human sperm.Design: Comparative study.Setting: Reproductive biology department.Patient(s): Nine sperm donors.Intervention(s): Sperm were exposed to interferon-α (IFN-α), IFN-γ, interleukin-12 (IL-12), Ca2+ ionophore (A23187), or progesterone under capacitating conditions.Main Outcome Measure(s): Cell lysates prepared from sperm and Jurkat T-cell line were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expression of JAKs (1–3 and TYK 2) and STATs (1–6) was examined by Western blot analysis. Effect of IFN-α, IFN-γ, IL-12, A23187, or progesterone on sperm STAT 1 or STAT 4 phosphorylation was determined by phospho-STAT 1 antibody or antiphosphotyrosine (APT) Western blot analysis. Indirect immunofluorescence and confocal laser scanning microscopy was used to confirm the specific staining of anti-TYK 2, anti-STAT 1, and anti-STAT 4 antibodies.Result(s): By Western blot analysis, only antibodies to TYK 2 of the JAK family, and antibodies to STAT 1 and STAT 4 members of the STAT family specifically recognized protein bands corresponding to TYK 2, STAT 1, and STAT 4 described in other cell types. By confocal microscopy, antibodies to TYK 2 reacted with the sperm tail as well as the apical region of sperm head, whereas antibodies to STAT 1 and STAT 4 reacted with the apical region of the sperm head. Tyrosine phosphorylation of STAT 1 in capacitated sperm was enhanced by IFN-α and IFN-γ, and that of STAT 4 was enhanced by IL-12. Both A23187 and progesterone markedly inhibited tyrosine phosphorylation of sperm STAT 4.Conclusion(s): Members of the JAK/STAT proteins, TYK 2, STAT 1, and STAT 4 are present and active in human sperm. The localization of STAT 1 and STAT 4 proteins to the apical region of the sperm head and their activation by IFN-α, IFN-γ, or IL-12 implicate a role for sperm STAT proteins in fertilization. We hypothesize that sperm-derived phosphorylated STAT 1 and STAT 4 could contribute to the pool of transcription factors during sperm–oocyte fusion as well as transmit signal to the oocyte nucleus. Therefore, defects in sperm TYK 2 and STAT 1- or STAT 4-mediated signaling pathway may have relevance to male factor infertility.