Variation among individual bulls in the distribution of acrosomal tyrosine-phosphorylated proteins in epididymal and ejaculated spermatozoa

Abstract

In Japanese black cattle, AI severely subfertile males have occasionally been found. In order to solve this problem, we previously asserted the need for exact examinations of acrosomal tyrosine-phosphorylated proteins and acrosome morphology in cryopreserved spermatozoa. In the present study, we further investigated acrosomal tyrosine-phosphorylated proteins in spermatozoa before cryopreservation and examined possible relationships between these phosphoproteins and acrosome stability. Ejaculated, epididymal and cryopreserved spermatozoa were subjected to examinations of general characteristics (motility, shape and acrosome morphology) and indirect immunofluorescence of acrosomal phosphoproteins. Unlike all general characteristic parameters, the distribution of acrosomal tyrosine-phosphorylated proteins in ejaculated and cauda epididymal spermatozoa varied considerably among bulls and was linked to the maintenance of morphologically normal acrosomes in cryopreserved spermatozoa or ejaculated spermatozoa after 270min incubation. Moreover, the distribution of these phosphoproteins was arranged in the spermatozoa of the proximal epididymides. These findings indicate that acrosomal tyrosine-phosphorylated proteins are distributionally arranged during early process of sperm maturation, that their distribution of cauda epididymal and ejaculated spermatozoa are largely different among bulls, and that varied states of acrosomal phosphoproteins may result in individual differences in acrosome stability among bulls.

Protein biomarkers for male artificial insemination subfertility in bovine spermatozoa

“…D, The distribution patterns of the SPACA1 proteins were observed in the immunostained epididymal spermatozoa 25,62 and the percentages of the spermatozoa that exhibited a mature (normal distribution) pattern of SPACA1 proteins were calculated, as described. 55 There were significant differences between the values with different letters (P<.05). C-Cap, central caput; D-Cap, distal caput; Cor, corpus; Cau, cauda indices were largely varied among the patients by between 13 and 199 points (full marks: 200 points).…”

“…Actually, the authors and their colleagues have been investigating the general characteristics and distribution patterns of acrosomal tyrosine-phosphorylated proteins (a protein biomarker for bull AI subfertility) in freshly ejaculated and cryopreserved spermatozoa from most of the sire candidates in the bovine AI center of Hyogo Prefecture, Japan. 26,55 However, it should be noted that a key point is to select suitable protein biomarkers for the sperm problems that happen in the AI centers because bull AI subfertility is caused by a deficiency or dysfunction of various molecules, including the regulatory proteins of ATP synthesis, acrosomal proteins, nuclear proteins, capacitation-related proteins, and seminal plasma proteins (Fig. 3).…”

“…Likewise, the tyrosinephosphorylated proteins were translocated to the acrosomal principal segment during bull sperm maturation. 55 In the head of bull (Japanese Black cattle) mature spermatozoa, ically normal acrosomes. 26,55 These results suggest that a lack of the acrosomal phosphoproteins (one of the main components is probably the 42 kDa tyrosine-phosphorylated SPACA1 protein) in ejaculated spermatozoa with normal general characteristics is linked to bull AI subfertility and lower sperm freezability.…”

“…55 In the head of bull (Japanese Black cattle) mature spermatozoa, ically normal acrosomes. 26,55 These results suggest that a lack of the acrosomal phosphoproteins (one of the main components is probably the 42 kDa tyrosine-phosphorylated SPACA1 protein) in ejaculated spermatozoa with normal general characteristics is linked to bull AI subfertility and lower sperm freezability. However, molecular analyses of these proteins are required for disclosing how the SPACA1 proteins can protect the acrosomes during the cryopreservation process and consequently maintain their sperm-fertilizing ability.…”

Characterization of extracellular Ca2+‐dependent full‐type hyperactivation in ejaculated boar spermatozoa preincubated with a cAMP analog

“…Indirect immunofluorescence was performed as described previously (Arai et al, ; Harayama, ; Harayama, Muroga, et al, ; Harayama, Sasaki, et al, ; Isono et al, ), with some modifications. Rabbit anti‐phosphorylated serine/threonine Protein kinase A substrate polyclonal antibody (1:100 dilution of item #9621) (Cell Signaling Technology), mouse anti‐phosphotyrosine monoclonal antibody (1:1,000 dilution of clone 4G10, item #05‐321) (Merck Millipore), fluorescein isothiocyanate (FITC)‐conjugated swine anti‐rabbit immunoglobulin polyclonal antibodies (1:100 dilution of item #F0205) (Dako), and FITC‐conjugated rabbit anti‐mouse immunoglobulin polyclonal antibodies (1:100 dilution of item #F0261) (Dako).…”