Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm

Abstract

The relationship between human sperm maturity and apoptosis is of interest because of the persistence of immature sperm in ejaculates in spite of various apoptotic processes during spermatogenesis. We assessed sperm maturity by HspA2 chaperone levels, and plasma membrane maturity by sperm binding to immobilized hyaluronic acid (HA). We also utilized objective morphometry. Sperm were stained with three antibody combinations: active caspase-3/creatine kinase (CK, a marker of cytoplasmic retention), caspase-3/the antiapoptotic Bcl-(XL), and CK/Bcl-(XL). In semen, 13% of sperm stained with CK, caspase-3 or Bcl-(XL), and 28% had stained with two markers. In the mature HA-bound sperm fraction, <4% were single- or double-stained. Regarding sperm regions, CK staining, whether alone or as double staining, occurred in the head and midpiece (15-20%), whereas caspase-3 and Bcl-(XL) were primarily (>80% of sperm) in the midpiece. Morphometrical attributes of clear, single- and double-stained sperm, in line with their more pronounced maturation arrest, showed an incremental increase in head size (due to cytoplasmic retention) and shorter tail length. We hypothesize that during faulty sperm development, three alternatives may occur: (i) elimination of aberrant germ cells by apoptosis; (ii) in surviving immature cells, caspase-3 is activated, and in response the antiapoptotic Bcl-(XL), and perhaps HspA2, provide protection; (iii) in a third type of immature sperm, in addition to the CK, caspase-3 and Bcl-(XL) expression, there are related manifestations of increased head size and shorter tail length. Thus, immature sperm may vary in the type of developmental arrest and in protection mechanisms for apoptosis. These variations are likely to explain the persistence of immature sperm in the ejaculate.

Impact of caspase activation in human spermatozoa

“…Particularly cytoplasmic droplets of immature sperm contain activated caspases supporting the theory of abortive apoptosis following incomplete spermatogenesis (Sakkas et al, 1999). In addition, the presence of the antiapoptotic regulator protein bcl-xL in mature sperm reduces caspase-3 activation (Cayli et al, 2004). Recent studies proved the decreased activity of caspase-3 in mature sperm by double probing using aniline blue and caspase-3 immunostaining on the same slide (Sati et al, 2008).…”

Mitochondria functionality and sperm quality

“…Although the extrinsic apoptotic pathway has been suggested to be active in sperm (Sakkas et al 1999), we will focus on the intrinsic (mitochondria-dependent) pathway, which involves, for example, both pro-and anti-apoptotic members of the Bcl family and especially on general apoptotic features, as there is clearly much more information at that level. In terms of the intrinsic pathway, anti-apoptotic Bcl-xL seems more prevalent in ejaculated abnormal/immature sperm, possibly as a spermatogenesis remnant (Cayli et al 2004), while the presence of both pro-and anti-apoptotic forms of Bcl-x have been proposed to exist in mature human sperm, but no correlations with sperm parameters were shown (Sakkas et al 2002).…”

Relationship between apoptotic markers in semen from fertile men and demographic, hormonal and seminal characteristics

“…Likewise, previous studies investigating pro-apoptotic proteins in the Bcl-2 family did not observe associations between Bcl and sperm concentration, morphology or motility. 12 However, Fas and Bcl-xL were positively correlated. Our observations support findings by Sakkas et al 14 with significant positive correlations between Fas and Bcl-x.…”

“…5 The theoretical framework of abortive apoptosis is based on the assumption that as germ cells progress into their terminal differentiation pathway, they become more and more transcriptionally and translationally silent and progressively lose their capacity to undergo programmed cell death in the form of classical apoptosis. Instead, a restricted form of apoptosis takes over, i.e., a sort of regulated cell death, 5 mirrored by the presence of nuclear DNA strand breaks in human ejaculated spermatozoa (as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay) and abnormal persistence of some proteins activated during the extrinsic and intrinsic apoptotic pathways, 8,12 such as the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL. In addition, other apoptotic hallmarks, such as caspase activation and phosphatidylserine exteriorization, have been detected in human spermatozoa.…”