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Biogenesis of sperm perinuclear theca and its role in sperm functional competence and fertilization
Journal of Reproductive Immunology 83 (2009) 2–7
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Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Biogenesis of sperm perinuclear theca and its role in sperm functional competence and fertilization Richard Oko a,∗ , Peter Sutovsky b,c a b c
Department of Anatomy and Cell Biology, Queen’s University, 9th Floor, Botterell Hall, Kingston, ON K7L 3N6, Canada Department of Animal Sciences, University of Missouri-Columbia, MO 65211-5300, USA Department of Obstetrics and Gynecology, University of Missouri-Columbia, MO 65211-5300, USA
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Article history: Received 16 December 2008 Accepted 27 May 2009 Keywords: Spermiogenesis Perinuclear theca Subacrosomal layer Acrosome formation Postacrosomal sheath Fertilization
a b s t r a c t The perinuclear theca is a condensed cytosolic protein layer that surrounds the mammalian sperm nucleus except in the region of tail implantation. It is resistant to non-ionic detergent extraction and makes up most of the cytosol of the sperm head. The perinuclear theca can be divided both structurally and compositionally into subacrosomal and postacrosomal regions. Most of the sequence-identities of investigated perinuclear theca proteins have been unexpected, revealing novel proteins as well as isoforms of somatic and conventionally nuclear proteins. Their characterizations have led us to propose that the perinuclear theca can be regionalized into two functional categories: the subacrosomal layer, involved in acrosomal assembly, and the postacrosomal sheath, involved in sperm–egg interactions during fertilization such as egg activation. Immunocytochemical investigation of the origins of well-characterized perinuclear theca proteins have led us to propose that: the subacrosomal layer is assembled relatively early in spermiogenesis from cytosolic proteins that “piggy-back” their way to the forming perinuclear theca on the periphery of acrosomic vesicles, while the postacrosomal sheath is assembled later, from cytosolic proteins that are transported up the microtubular manchette as it descends over the caudal half of the elongating spermatid nucleus. In this review data collected on resident perinuclear theca proteins, SubH2Bv, RAB2, PAWP and the four core somatic histones, is used to substantiate these hypotheses. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The specialized structure of mammalian spermatozoa reflects a complex process of biochemical transformation, which takes place mainly during the haploid phase of germ cell development termed spermiogenesis (Figs. 1 and 2). With the exception of mitochondria none of the standard cytoplasmic organelles such as endoplasmic reticulum (ER), Golgi apparatus and centrioles are seen in the mature mammalian spermatozoa. Instead, what is observed in the spermatozoon are derivatives of these organelles, such as
∗ Corresponding author. Tel.: +1 613 533 2858; fax: +1 613 533 2566. E-mail address: [email protected] (R. Oko). 0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2009.05.008
the acrosome formed by the ER–Golgi system (Clermont et al., 1993), the transient Golgi-derived saccular elements (Oko et al., 1993), the axoneme derived from one of the two centrioles (Oko and Clermont, 1991) and a very condensed nucleus (Oko et al., 1996). In addition to these transformations, some of the most conspicuous developmental changes are concerned with the elaboration of specialized “cytoskeletal” head and tail components of which there appear to be no equivalent in any other cell type (Oko and Morales, 1996; Oko, 1998). In the sperm head the major cytoskeletal component is the perinuclear theca (PT), which is not composed of traditional cytoskeletal proteins as was originally thought, but rather from a variety of cytosolic and nuclear proteins. Our objectives have been to sequence identify and characterize the PT proteins with the
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Fig. 1. The route proposed for the assembly of subacrosomal layer (SAL) proteins (orange dots) of the perinuclear theca (PT) during the early steps of bovine spermiogenesis. The Golgi phase of spermiogenesis is represented by steps 2 and 3 spermatids, the cap phase is represented by steps 4 and 7 spermatids and the early elongating phase is represented by a step 8 spermatid. SAL–PT proteins associate with the cytosolic side of the acrosomal membrane during the Golgi and cap phases of spermiogenesis. Towards the end of the cap phase and the beginning of spermatid elongation, SAL proteins disappear from the surface of the outer acrosomal membrane but are retained over the equatorial segment region of this membrane and beneath the inner acrosomal membrane, where they remain permanently as the SAL part of the PT (coloured in solid orange). The nucleus is coloured in blue. PA, proacrosomic granules; AV, acrosomic vesicle; GA, Golgi apparatus; AC, acrosomic cap; MAN, manchette; Cl, cytoplasmic lobe. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. The route proposed for the assembly of postacrosomal sheath (PAS) proteins (red dots) of the perinuclear theca (PT) during the elongation phase of bovine spermiogenesis. Spermatid steps 11–14 are depicted, with step 14 representing a mid-sagital sectional view of a structurally mature spermatid or spermatozoa. The changing light to dark shade of blue represents the condensation of the nucleus, which is occurring in steps 11 and 12 spermatids concomitant with the descent of the microtubular manchette (MAN). The microtubules (MT) of the manchette disintegrate in step 13. The manchette serves as a storage site and transportation track for PAS proteins, which are translated in the cytoplasmic lobe (CL). Upon manchette descent in steps 11 and 12, manchette associated PAS proteins are translocated to the forming PAS, most likely facilitated by microtubule motor proteins. Initially some of the newly transported PAS proteins share space with the subacrosomal layer (SAL) in the equatorial segment region (ESR) but by step 14 with the thinning of the equatorial segment (ES) become confined to the PAS region (solid red) of the PT. NR, nuclear ring of the manchette; A, acrosome; CP, connecting piece of the sperm tail. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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intention of resolving the developmental pathways leading to the assembly of the different regions of the PT and elucidating their roles in spermiogenesis and fertilization. 2. Perinuclear theca composition The PT is a detergent resistant capsule that surrounds the mature sperm nucleus, except in the tail implantation region (Fig. 2, step 14 spermatid or spermatozoa). Apically, PT resides between the inner acrosomal membrane and the nuclear envelope making up the subacrosomal layer (SAL), while caudally, it resides between plasmalemma and the nuclear envelope making up the postacrosomal sheath (PAS) (Oko and Maravei, 1994; Oko, 1995). The SAL part of the PT is also structurally and compositionally continuous with the part of the outer periacrosomal layer, which is sandwiched between the outer acrosomal membrane and plasmalemma in the equatorial region (Oko et al., 1990; Oko and Maravei, 1994). The structural continuity of the three regions of the PT is retained after non-ionic detergent extraction of sonicated and isolated sperm heads (Oko and Maravei, 1994; Oko et al., 2001; Mountjoy et al., 2008). Essentially, all that remains is a shell of PT material surrounding the condensed nucleus. This head fraction serves as an ideal starting point for extracting the PT proteins. Sequential extraction steps in 1 M NaCl and 100 mM NaOH completely remove the PT from the nucleus and allow their resolution on SDS-PAGE. Antibodies raised against the whole NaCl (high salt) and NaOH (alkaline) extracts only immunolabel the PT. To date, by a variety of proteomic approaches, we have sequence identified and raised specific antibodies to a multitude of PT proteins (Oko and Morales, 1994; Oko and Maravei, 1994; Korley et al., 1997; Oko et al., 2001; Aul and Oko, 2002; Tovich and Oko, 2003; Wu et al., 2007a; Mountjoy et al., 2008). Together with Drs. Franke and Heid, who have also contributed significantly to the composition of the PT (Hess et al., 1993; Hess et al., 1995; von Bulow et al., 1995; von Bulow et al., 1997; Heid et al., 2002), we are utilizing specific antibodies against PT proteins to resolve the pathways leading to the assembly of the different regions of the PT during spermiogenesis. 3. SAL–PT assembly during acrosome formation and function of SAL constituents in acrosomal biogenesis The formation of the acrosome during the first half of spermiogenesis can be divided into Golgi and cap phases, distinguished by two different phases of secretory activity by the Golgi apparatus (Tang et al., 1982; Thorne-Tjomsland et al., 1988; Clermont et al., 1993; Oko and Clermont, 1998) (Fig. 1). In the first phase (steps 1–3) the Golgi secretes several small and dense secretory granules rich in hydrolytic enzymes such as proacrosin. These proacrosomic granules coalesce to form a single larger, dense cored, spherical acrosomic vesicle, which associates with the nuclear envelope via a proteinaceous layer referred to as the PT. In the second phase (steps 4–7) the acrosomic vesicle enlarges by fusing with numerous small carrier vesicles originating from the trans face of the Golgi apparatus. As a result, there is a continual addition of
membrane and glycoprotein, allowing the less dense cortex part of the acrosomic vesicle to expand over the nucleus and take the shape of a cap. It should be emphasized that at the ultrastructural level the expansion of the acrosomic system is synchronous with the expansion of the underlying PT. Therefore, it would be presumptive to conclude that the PT is a precursor to acrosomal-nuclear docking (in other words, is on the surface of the nuclear envelope before acrosomal membrane attachment), a concept recently proposed by Kierszenbaum and co-workers (Tres and Kierszenbaum, 1996; Kierszenbaum et al., 2003). These investigators made no reference to our earlier studies (Oko and Maravei, 1995; Oko, 1995; Aul and Oko, 2002) in which we provided evidence that SAL proteins get to their subacrosomal destination by coating the proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis (Fig. 1). Utilizing immune sera against the alkaline PT extract, in which the majority of PT proteins were found, we showed that PT-immunogold labelling was associated with the entire acrosomal membrane of the acrosomic vesicle before its attachment to the nuclear envelope. As no PT antigenic sites were associated with the nuclear envelope before acrosomic vesicle attachment, we suggested that the peripheral association of these cytosolic proteins with the membrane of the acrosomic vesicle is an important requirement for the recognition of nuclear attachment. More recently utilizing a variety of proteomic approaches, we sequence identified several prominent PT proteins that are exclusively localized to SAL of which two, SubH2Bv and RAB2A, are well characterized (Aul and Oko, 2002; Mountjoy et al., 2008). Their sequence-identities allowed us to produce or procure antibodies against specific epitopes. The cell localization data obtained with these SAL-protein specific antibodies, together with PASprotein specific antibodies (see below), reinforced the idea that the PT is indeed compartmentalized into two regions. More importantly the tissue localization of these anti-SAL specific antibodies confirmed our hypothesis that SAL is assembled from cytosolic proteins that “piggy-back” on the periphery of emerging acrosomic vesicles, indicative of their involvement in acrosomal formation (Fig. 1). Interestingly the properties of these two prominent SAL proteins are unrelated but yet they both employ the same developmental route to become the subacrosomal part of the PT. RAB2A belongs to the RAB subgroup of the Ras superfamily. The RAB family members have emerged as essential regulators of vesicular transport including vesicle formation, actin- and tubulin-dependent vesicle movement and targeting to and fusion with membranes (Pereira-Leal and Seabra, 2000, 2001; Stenmark and Olkkonen, 2001). The localization of RAB2A on the acrosomal membrane during acrosomal formation implies that its function may be to regulate the transport and fusion of small secretory vesicles to and with the growing proacrosomic vesicles and acrosomic vesicle. SubH2Bv on the other hand is a histone H2B variant that would be expected to reside in the sperm nucleus, rather than the cytosol. Ironically, a feature making it unique from H2B is the presence of a bipartite nuclear localization signal
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(NLS) (Aul and Oko, 2002). By performing transfection studies in somatic cells we have shown that SubH2Bv’s bipartite NLS directs it or GFP into the somatic cell nucleus (Aul, Tran and Oko, unpublished data, Ph.D. Thesis of Aul). Since SubH2Bv is never found within the spermatid nucleus we hypothesized that its NLS targets the SubH2Bv coated acrosomic vesicle to the spermatid nuclear envelope, which at this crucial stage of acrosomal-nuclear docking is devoid of nuclear pores. In order to target substrates to the nucleus, all bipartite NLS are first required to bind with importin ␣, which then binds to importin  to form a trimeric complex in the cytoplasm (Gorlich et al., 1996; Cingolani et al., 1999). Such a requirement implies that the importins would have to interact with SubH2Bv to bring the SubH2Bv coated acrosomic vesicle to the nucleus. This concept has recently been supported in our lab by localization of importin ␣ around the proacrosomic and acrosomic vesicles and in the subacrosomal space of the forming acrosome (Fig. 1). Furthermore, extraction of importin ␣ and importin  from isolated sperm heads and round and elongated spermatids as well as their co-precipitation with SubH2B in cellular pull down assays (Tran, Aul and Oko, unpublished data, M.Sc. Thesis of Tran) fortify this novel hypothesis that acrosomal-nuclear docking is a process dependent upon the classical bipartite NLS-substrate/importin ␣/importin  interaction. In summary then we predict that most proteins occupying the subacrosomal region of the mature PT are vestiges of functions related either to secretory vesicular formation, fusion, transport, nuclear docking or acrosomal capping. For SAL proteins to perform such roles their most advantageous position during development would indeed be association with the acrosomal membrane of growing proacrosomic and acrosomic secretory vesicles. Once acrosomal biogenesis is over SAL–PT proteins become trapped between the inner acrosomal membrane and nuclear envelope, however, in this position they are well situated to be part of a protein complex that binds the mature acrosome firmly to the nucleus. 4. PAS-PT formation during microtubular manchette descent and role of PAS constituents in fertilization Just as the emergence of SAL appears to occur in concert with acrosome development, PAS appears to be assembled during the caudal descent of the microtubular manchette, concomitant with spermatid elongation (Barth and Oko, 1989; Oko, 1998). The manchette, a microtubular network forming a girdle-like structure around the caudal half of the elongating spermatid nucleus, extends deep into the distal cytoplasm where all the proteins involved in spermatid elongation are expressed (Fig. 2, step 11). It is a transient structure, thought to be involved with spermatid-head shaping (Meistrich et al., 1990; Russell et al., 1991) and “intra-manchette transport” of proteins (Kierszenbaum et al., 2002; Kierszenbaum, 2002). We provided the first evidence for intra-manchette transport by detecting a population of PAS-resident somatic core histones on the manchette just before it descends down the spermatid nucleus (Tovich et al., 2004). These histones are
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then deposited adjacent to the caudal half of the spermatid nucleus, to form part of the PAS, in the wake of manchette descent (Tovich et al., 2004) (Fig. 2, steps 11 and 12). However, in the case of the core somatic histones (H3, H2B, H2A and H4) we were not able to clearly demonstrate that they are synthesized de novo in the distal cytoplasm of elongating spermatids raising the possibility of histone recycling from the spermatid nucleus to the manchette before their PAS deposition. Therefore, to confirm our hypothesis that the assembly of de novo synthesized PAS proteins is dependent on intra-manchette protein transport as well as manchette descent we followed the developmental sequence of a PAS-resident protein, PAWP, which is synthesized de novo in the cytoplasmic lobe of elongating spermatids (Wu et al., 2007a). Utilizing specific antibodies generated against PAWP and tubulin, we have co-localized these two proteins by immunofluorescence on the manchette in testicular spreads and shown their divergence as the manchette slides down the spermatid nucleus, leaving behind the PAWP packaged PAS (Fig. 2) (Wu et al., 2007b). Along with immunofluorescence we have analyzed PAWP-PAS assembly ultrastructurally by immunogold immunocytochemistry and confirmed the assembly of PAS proteins as depicted in the above figure. We predict that the assembly of most PAS (calyx) proteins will follow the same pattern. To address the hypothesis, that PAS assembly is independent of SAL formation, ultrastructural analysis as well as PAS-protein immunolocalization was performed on testicular sections of NB-DNJ treated mice who fail to form a sperm acrosome and subacrosomal layer (van der Spoel et al., 2002; Walden et al., 2006) In favour of this hypothesis, it was found that the PAS is normally assembled in these affected mice (Wu et al., 2007b). Interestingly, the acrosome-less sperm of these mice when used in ICSI still retained egg-activating ability (Suganuma et al., 2005) reinforcing the hypothesis that the PAS is the region of the PT in which the sperm–oocyte activating factor (Sutovsky et al., 1996; Sutovsky et al., 1997; Sutovsky et al., 2003). It was observed that local solubilization of the PAS part of the PT was sufficient to elicit full oocyte activation during IVF (Sutovsky et al., 2003). In search of the sperm–oocyte activating factor candidate within the PAS of the PT we basically did a proteomic search of the alkaline PT extract, the sperm fraction in which egg-activating ability resides. Of all the PT proteins that we sequence identified, only one so far has the characteristic of a signal transduction protein. Because this protein resides exclusively in the PAS and contains a functional PPXY consensus binding site for group I WW domain containing proteins we termed it PAWP, which turned out to be an appropriate name as microinjection of its recombinant form was able to activate mammalian as well as amphibian oocytes (Wu et al., 2007a). We were able to show that PAWP is brought in with the sperm head into the oocyte cytoplasm during fertilization and that oocytes microinjected with sperm containing specific inhibitors of PAWP are unable to undergo meiotic resumption and pronuclei formation (Wu et al., 2007a). These two criteria are essential for establishing a protein as a sperm–oocyte activating factor candidate, and as far as we are aware have
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not yet been shown for other proposed sperm–oocyte activating factors. Most recently, utilizing the frog as our model we were able to substantiate (Qin, Aarabi, Oko, unpublished data, M.Sc. Thesis of Qin) that PAWP signalling within the oocyte is upstream of calcium release, which is considered to be the universal signalling mechanism for the resumption of the oocyte cell cycle during fertilization. Presently we are confirming PAWP’s upstream regulatory role in calcium signalling in mammalian oocytes. In summary then we predict that most proteins found in the PAS-PT are synthesized in the cytoplasmic lobe of elongating spermatids and transported up the microtubular manchette for final deposition and assembly in the postacrosomal region. It is important to note that filling this region before PAS-protein deposition and assembly are the microtubules of the manchette, which are squeezed between the plasmalemma and the nuclear envelope of the condensing spermatid nucleus (see Fig. 2, step 11). Therefore, in order to deposit PAS proteins that are stored on the microtubules and have them assemble into the PT structure, it is considered compulsory for the manchette to migrate distally into the cytoplasmic lobe. In its wake it deposits the proteins with the result of filling the space it once occupied, in the caudal region of the spermatid head. Another observation we have made is that so far all the PAS proteins that we have identified and characterized appear to participate in sperm–egg interactions during fertilization. PAWP as we discussed is involved in egg activation. The PAS contingent of core somatic histones, on the other hand, like PAWP dissolve in the oocyte cytoplasm right after sperm entry and appear to immediately surround and infiltrate the decondensing male nucleus (Tovich, Sutovsky, Oko, unpublished data, Ph.D. Thesis of Tovich). We suggest that these core somatic histones would be essential in stabilizing the chromatin and preventing it from degradation once it is released from its condensed state by the reduction of disulfide bonds between protamines. These observations lead us to speculate that the main purpose for the formation of the PAS is to assist the sperm in intra-cytoplasmic aspects of oocyte fertilization. Acknowledgement Supported by a grant (#MOP-84440) from the Canadian Institute of Health Research (R.O.). P.S. was supported by National Research Initiative Competitive Grant number 2007-01319 from the USDA Cooperative State Research, Education and Extension Service and seed funding from the Food for the 21st Century Program of the University of Missouri-Columbia. References Aul, R.B., Oko, R., 2002. The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis. Dev. Biol. 242, 376–387. Barth, A.D., Oko, R., 1989. Normal bovine spermatogenesis and sperm maturation. In: Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press, Ames, pp. 19–88. Cingolani, G., Petosa, C., Weis, K., Muller, C.W., 1999. Structure of importinbeta bound to the IBB domain of importin-alpha. Nature 399, 221–229. Clermont, Y., Oko, R., Hermo, L., 1993. Cell and molecular biology of the testis. In: Desjardins, C., Ewing, L. (Eds.), Cell Biology of
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Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Biogenesis of sperm perinuclear theca and its role in sperm functional competence and fertilization Richard Oko a,∗ , Peter Sutovsky b,c a b c
Department of Anatomy and Cell Biology, Queen’s University, 9th Floor, Botterell Hall, Kingston, ON K7L 3N6, Canada Department of Animal Sciences, University of Missouri-Columbia, MO 65211-5300, USA Department of Obstetrics and Gynecology, University of Missouri-Columbia, MO 65211-5300, USA
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Article history: Received 16 December 2008 Accepted 27 May 2009 Keywords: Spermiogenesis Perinuclear theca Subacrosomal layer Acrosome formation Postacrosomal sheath Fertilization
a b s t r a c t The perinuclear theca is a condensed cytosolic protein layer that surrounds the mammalian sperm nucleus except in the region of tail implantation. It is resistant to non-ionic detergent extraction and makes up most of the cytosol of the sperm head. The perinuclear theca can be divided both structurally and compositionally into subacrosomal and postacrosomal regions. Most of the sequence-identities of investigated perinuclear theca proteins have been unexpected, revealing novel proteins as well as isoforms of somatic and conventionally nuclear proteins. Their characterizations have led us to propose that the perinuclear theca can be regionalized into two functional categories: the subacrosomal layer, involved in acrosomal assembly, and the postacrosomal sheath, involved in sperm–egg interactions during fertilization such as egg activation. Immunocytochemical investigation of the origins of well-characterized perinuclear theca proteins have led us to propose that: the subacrosomal layer is assembled relatively early in spermiogenesis from cytosolic proteins that “piggy-back” their way to the forming perinuclear theca on the periphery of acrosomic vesicles, while the postacrosomal sheath is assembled later, from cytosolic proteins that are transported up the microtubular manchette as it descends over the caudal half of the elongating spermatid nucleus. In this review data collected on resident perinuclear theca proteins, SubH2Bv, RAB2, PAWP and the four core somatic histones, is used to substantiate these hypotheses. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The specialized structure of mammalian spermatozoa reflects a complex process of biochemical transformation, which takes place mainly during the haploid phase of germ cell development termed spermiogenesis (Figs. 1 and 2). With the exception of mitochondria none of the standard cytoplasmic organelles such as endoplasmic reticulum (ER), Golgi apparatus and centrioles are seen in the mature mammalian spermatozoa. Instead, what is observed in the spermatozoon are derivatives of these organelles, such as
∗ Corresponding author. Tel.: +1 613 533 2858; fax: +1 613 533 2566. E-mail address: [email protected] (R. Oko). 0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2009.05.008
the acrosome formed by the ER–Golgi system (Clermont et al., 1993), the transient Golgi-derived saccular elements (Oko et al., 1993), the axoneme derived from one of the two centrioles (Oko and Clermont, 1991) and a very condensed nucleus (Oko et al., 1996). In addition to these transformations, some of the most conspicuous developmental changes are concerned with the elaboration of specialized “cytoskeletal” head and tail components of which there appear to be no equivalent in any other cell type (Oko and Morales, 1996; Oko, 1998). In the sperm head the major cytoskeletal component is the perinuclear theca (PT), which is not composed of traditional cytoskeletal proteins as was originally thought, but rather from a variety of cytosolic and nuclear proteins. Our objectives have been to sequence identify and characterize the PT proteins with the
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Fig. 1. The route proposed for the assembly of subacrosomal layer (SAL) proteins (orange dots) of the perinuclear theca (PT) during the early steps of bovine spermiogenesis. The Golgi phase of spermiogenesis is represented by steps 2 and 3 spermatids, the cap phase is represented by steps 4 and 7 spermatids and the early elongating phase is represented by a step 8 spermatid. SAL–PT proteins associate with the cytosolic side of the acrosomal membrane during the Golgi and cap phases of spermiogenesis. Towards the end of the cap phase and the beginning of spermatid elongation, SAL proteins disappear from the surface of the outer acrosomal membrane but are retained over the equatorial segment region of this membrane and beneath the inner acrosomal membrane, where they remain permanently as the SAL part of the PT (coloured in solid orange). The nucleus is coloured in blue. PA, proacrosomic granules; AV, acrosomic vesicle; GA, Golgi apparatus; AC, acrosomic cap; MAN, manchette; Cl, cytoplasmic lobe. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. The route proposed for the assembly of postacrosomal sheath (PAS) proteins (red dots) of the perinuclear theca (PT) during the elongation phase of bovine spermiogenesis. Spermatid steps 11–14 are depicted, with step 14 representing a mid-sagital sectional view of a structurally mature spermatid or spermatozoa. The changing light to dark shade of blue represents the condensation of the nucleus, which is occurring in steps 11 and 12 spermatids concomitant with the descent of the microtubular manchette (MAN). The microtubules (MT) of the manchette disintegrate in step 13. The manchette serves as a storage site and transportation track for PAS proteins, which are translated in the cytoplasmic lobe (CL). Upon manchette descent in steps 11 and 12, manchette associated PAS proteins are translocated to the forming PAS, most likely facilitated by microtubule motor proteins. Initially some of the newly transported PAS proteins share space with the subacrosomal layer (SAL) in the equatorial segment region (ESR) but by step 14 with the thinning of the equatorial segment (ES) become confined to the PAS region (solid red) of the PT. NR, nuclear ring of the manchette; A, acrosome; CP, connecting piece of the sperm tail. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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intention of resolving the developmental pathways leading to the assembly of the different regions of the PT and elucidating their roles in spermiogenesis and fertilization. 2. Perinuclear theca composition The PT is a detergent resistant capsule that surrounds the mature sperm nucleus, except in the tail implantation region (Fig. 2, step 14 spermatid or spermatozoa). Apically, PT resides between the inner acrosomal membrane and the nuclear envelope making up the subacrosomal layer (SAL), while caudally, it resides between plasmalemma and the nuclear envelope making up the postacrosomal sheath (PAS) (Oko and Maravei, 1994; Oko, 1995). The SAL part of the PT is also structurally and compositionally continuous with the part of the outer periacrosomal layer, which is sandwiched between the outer acrosomal membrane and plasmalemma in the equatorial region (Oko et al., 1990; Oko and Maravei, 1994). The structural continuity of the three regions of the PT is retained after non-ionic detergent extraction of sonicated and isolated sperm heads (Oko and Maravei, 1994; Oko et al., 2001; Mountjoy et al., 2008). Essentially, all that remains is a shell of PT material surrounding the condensed nucleus. This head fraction serves as an ideal starting point for extracting the PT proteins. Sequential extraction steps in 1 M NaCl and 100 mM NaOH completely remove the PT from the nucleus and allow their resolution on SDS-PAGE. Antibodies raised against the whole NaCl (high salt) and NaOH (alkaline) extracts only immunolabel the PT. To date, by a variety of proteomic approaches, we have sequence identified and raised specific antibodies to a multitude of PT proteins (Oko and Morales, 1994; Oko and Maravei, 1994; Korley et al., 1997; Oko et al., 2001; Aul and Oko, 2002; Tovich and Oko, 2003; Wu et al., 2007a; Mountjoy et al., 2008). Together with Drs. Franke and Heid, who have also contributed significantly to the composition of the PT (Hess et al., 1993; Hess et al., 1995; von Bulow et al., 1995; von Bulow et al., 1997; Heid et al., 2002), we are utilizing specific antibodies against PT proteins to resolve the pathways leading to the assembly of the different regions of the PT during spermiogenesis. 3. SAL–PT assembly during acrosome formation and function of SAL constituents in acrosomal biogenesis The formation of the acrosome during the first half of spermiogenesis can be divided into Golgi and cap phases, distinguished by two different phases of secretory activity by the Golgi apparatus (Tang et al., 1982; Thorne-Tjomsland et al., 1988; Clermont et al., 1993; Oko and Clermont, 1998) (Fig. 1). In the first phase (steps 1–3) the Golgi secretes several small and dense secretory granules rich in hydrolytic enzymes such as proacrosin. These proacrosomic granules coalesce to form a single larger, dense cored, spherical acrosomic vesicle, which associates with the nuclear envelope via a proteinaceous layer referred to as the PT. In the second phase (steps 4–7) the acrosomic vesicle enlarges by fusing with numerous small carrier vesicles originating from the trans face of the Golgi apparatus. As a result, there is a continual addition of
membrane and glycoprotein, allowing the less dense cortex part of the acrosomic vesicle to expand over the nucleus and take the shape of a cap. It should be emphasized that at the ultrastructural level the expansion of the acrosomic system is synchronous with the expansion of the underlying PT. Therefore, it would be presumptive to conclude that the PT is a precursor to acrosomal-nuclear docking (in other words, is on the surface of the nuclear envelope before acrosomal membrane attachment), a concept recently proposed by Kierszenbaum and co-workers (Tres and Kierszenbaum, 1996; Kierszenbaum et al., 2003). These investigators made no reference to our earlier studies (Oko and Maravei, 1995; Oko, 1995; Aul and Oko, 2002) in which we provided evidence that SAL proteins get to their subacrosomal destination by coating the proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis (Fig. 1). Utilizing immune sera against the alkaline PT extract, in which the majority of PT proteins were found, we showed that PT-immunogold labelling was associated with the entire acrosomal membrane of the acrosomic vesicle before its attachment to the nuclear envelope. As no PT antigenic sites were associated with the nuclear envelope before acrosomic vesicle attachment, we suggested that the peripheral association of these cytosolic proteins with the membrane of the acrosomic vesicle is an important requirement for the recognition of nuclear attachment. More recently utilizing a variety of proteomic approaches, we sequence identified several prominent PT proteins that are exclusively localized to SAL of which two, SubH2Bv and RAB2A, are well characterized (Aul and Oko, 2002; Mountjoy et al., 2008). Their sequence-identities allowed us to produce or procure antibodies against specific epitopes. The cell localization data obtained with these SAL-protein specific antibodies, together with PASprotein specific antibodies (see below), reinforced the idea that the PT is indeed compartmentalized into two regions. More importantly the tissue localization of these anti-SAL specific antibodies confirmed our hypothesis that SAL is assembled from cytosolic proteins that “piggy-back” on the periphery of emerging acrosomic vesicles, indicative of their involvement in acrosomal formation (Fig. 1). Interestingly the properties of these two prominent SAL proteins are unrelated but yet they both employ the same developmental route to become the subacrosomal part of the PT. RAB2A belongs to the RAB subgroup of the Ras superfamily. The RAB family members have emerged as essential regulators of vesicular transport including vesicle formation, actin- and tubulin-dependent vesicle movement and targeting to and fusion with membranes (Pereira-Leal and Seabra, 2000, 2001; Stenmark and Olkkonen, 2001). The localization of RAB2A on the acrosomal membrane during acrosomal formation implies that its function may be to regulate the transport and fusion of small secretory vesicles to and with the growing proacrosomic vesicles and acrosomic vesicle. SubH2Bv on the other hand is a histone H2B variant that would be expected to reside in the sperm nucleus, rather than the cytosol. Ironically, a feature making it unique from H2B is the presence of a bipartite nuclear localization signal
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(NLS) (Aul and Oko, 2002). By performing transfection studies in somatic cells we have shown that SubH2Bv’s bipartite NLS directs it or GFP into the somatic cell nucleus (Aul, Tran and Oko, unpublished data, Ph.D. Thesis of Aul). Since SubH2Bv is never found within the spermatid nucleus we hypothesized that its NLS targets the SubH2Bv coated acrosomic vesicle to the spermatid nuclear envelope, which at this crucial stage of acrosomal-nuclear docking is devoid of nuclear pores. In order to target substrates to the nucleus, all bipartite NLS are first required to bind with importin ␣, which then binds to importin  to form a trimeric complex in the cytoplasm (Gorlich et al., 1996; Cingolani et al., 1999). Such a requirement implies that the importins would have to interact with SubH2Bv to bring the SubH2Bv coated acrosomic vesicle to the nucleus. This concept has recently been supported in our lab by localization of importin ␣ around the proacrosomic and acrosomic vesicles and in the subacrosomal space of the forming acrosome (Fig. 1). Furthermore, extraction of importin ␣ and importin  from isolated sperm heads and round and elongated spermatids as well as their co-precipitation with SubH2B in cellular pull down assays (Tran, Aul and Oko, unpublished data, M.Sc. Thesis of Tran) fortify this novel hypothesis that acrosomal-nuclear docking is a process dependent upon the classical bipartite NLS-substrate/importin ␣/importin  interaction. In summary then we predict that most proteins occupying the subacrosomal region of the mature PT are vestiges of functions related either to secretory vesicular formation, fusion, transport, nuclear docking or acrosomal capping. For SAL proteins to perform such roles their most advantageous position during development would indeed be association with the acrosomal membrane of growing proacrosomic and acrosomic secretory vesicles. Once acrosomal biogenesis is over SAL–PT proteins become trapped between the inner acrosomal membrane and nuclear envelope, however, in this position they are well situated to be part of a protein complex that binds the mature acrosome firmly to the nucleus. 4. PAS-PT formation during microtubular manchette descent and role of PAS constituents in fertilization Just as the emergence of SAL appears to occur in concert with acrosome development, PAS appears to be assembled during the caudal descent of the microtubular manchette, concomitant with spermatid elongation (Barth and Oko, 1989; Oko, 1998). The manchette, a microtubular network forming a girdle-like structure around the caudal half of the elongating spermatid nucleus, extends deep into the distal cytoplasm where all the proteins involved in spermatid elongation are expressed (Fig. 2, step 11). It is a transient structure, thought to be involved with spermatid-head shaping (Meistrich et al., 1990; Russell et al., 1991) and “intra-manchette transport” of proteins (Kierszenbaum et al., 2002; Kierszenbaum, 2002). We provided the first evidence for intra-manchette transport by detecting a population of PAS-resident somatic core histones on the manchette just before it descends down the spermatid nucleus (Tovich et al., 2004). These histones are
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then deposited adjacent to the caudal half of the spermatid nucleus, to form part of the PAS, in the wake of manchette descent (Tovich et al., 2004) (Fig. 2, steps 11 and 12). However, in the case of the core somatic histones (H3, H2B, H2A and H4) we were not able to clearly demonstrate that they are synthesized de novo in the distal cytoplasm of elongating spermatids raising the possibility of histone recycling from the spermatid nucleus to the manchette before their PAS deposition. Therefore, to confirm our hypothesis that the assembly of de novo synthesized PAS proteins is dependent on intra-manchette protein transport as well as manchette descent we followed the developmental sequence of a PAS-resident protein, PAWP, which is synthesized de novo in the cytoplasmic lobe of elongating spermatids (Wu et al., 2007a). Utilizing specific antibodies generated against PAWP and tubulin, we have co-localized these two proteins by immunofluorescence on the manchette in testicular spreads and shown their divergence as the manchette slides down the spermatid nucleus, leaving behind the PAWP packaged PAS (Fig. 2) (Wu et al., 2007b). Along with immunofluorescence we have analyzed PAWP-PAS assembly ultrastructurally by immunogold immunocytochemistry and confirmed the assembly of PAS proteins as depicted in the above figure. We predict that the assembly of most PAS (calyx) proteins will follow the same pattern. To address the hypothesis, that PAS assembly is independent of SAL formation, ultrastructural analysis as well as PAS-protein immunolocalization was performed on testicular sections of NB-DNJ treated mice who fail to form a sperm acrosome and subacrosomal layer (van der Spoel et al., 2002; Walden et al., 2006) In favour of this hypothesis, it was found that the PAS is normally assembled in these affected mice (Wu et al., 2007b). Interestingly, the acrosome-less sperm of these mice when used in ICSI still retained egg-activating ability (Suganuma et al., 2005) reinforcing the hypothesis that the PAS is the region of the PT in which the sperm–oocyte activating factor (Sutovsky et al., 1996; Sutovsky et al., 1997; Sutovsky et al., 2003). It was observed that local solubilization of the PAS part of the PT was sufficient to elicit full oocyte activation during IVF (Sutovsky et al., 2003). In search of the sperm–oocyte activating factor candidate within the PAS of the PT we basically did a proteomic search of the alkaline PT extract, the sperm fraction in which egg-activating ability resides. Of all the PT proteins that we sequence identified, only one so far has the characteristic of a signal transduction protein. Because this protein resides exclusively in the PAS and contains a functional PPXY consensus binding site for group I WW domain containing proteins we termed it PAWP, which turned out to be an appropriate name as microinjection of its recombinant form was able to activate mammalian as well as amphibian oocytes (Wu et al., 2007a). We were able to show that PAWP is brought in with the sperm head into the oocyte cytoplasm during fertilization and that oocytes microinjected with sperm containing specific inhibitors of PAWP are unable to undergo meiotic resumption and pronuclei formation (Wu et al., 2007a). These two criteria are essential for establishing a protein as a sperm–oocyte activating factor candidate, and as far as we are aware have
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not yet been shown for other proposed sperm–oocyte activating factors. Most recently, utilizing the frog as our model we were able to substantiate (Qin, Aarabi, Oko, unpublished data, M.Sc. Thesis of Qin) that PAWP signalling within the oocyte is upstream of calcium release, which is considered to be the universal signalling mechanism for the resumption of the oocyte cell cycle during fertilization. Presently we are confirming PAWP’s upstream regulatory role in calcium signalling in mammalian oocytes. In summary then we predict that most proteins found in the PAS-PT are synthesized in the cytoplasmic lobe of elongating spermatids and transported up the microtubular manchette for final deposition and assembly in the postacrosomal region. It is important to note that filling this region before PAS-protein deposition and assembly are the microtubules of the manchette, which are squeezed between the plasmalemma and the nuclear envelope of the condensing spermatid nucleus (see Fig. 2, step 11). Therefore, in order to deposit PAS proteins that are stored on the microtubules and have them assemble into the PT structure, it is considered compulsory for the manchette to migrate distally into the cytoplasmic lobe. In its wake it deposits the proteins with the result of filling the space it once occupied, in the caudal region of the spermatid head. Another observation we have made is that so far all the PAS proteins that we have identified and characterized appear to participate in sperm–egg interactions during fertilization. PAWP as we discussed is involved in egg activation. The PAS contingent of core somatic histones, on the other hand, like PAWP dissolve in the oocyte cytoplasm right after sperm entry and appear to immediately surround and infiltrate the decondensing male nucleus (Tovich, Sutovsky, Oko, unpublished data, Ph.D. Thesis of Tovich). We suggest that these core somatic histones would be essential in stabilizing the chromatin and preventing it from degradation once it is released from its condensed state by the reduction of disulfide bonds between protamines. These observations lead us to speculate that the main purpose for the formation of the PAS is to assist the sperm in intra-cytoplasmic aspects of oocyte fertilization. Acknowledgement Supported by a grant (#MOP-84440) from the Canadian Institute of Health Research (R.O.). P.S. was supported by National Research Initiative Competitive Grant number 2007-01319 from the USDA Cooperative State Research, Education and Extension Service and seed funding from the Food for the 21st Century Program of the University of Missouri-Columbia. References Aul, R.B., Oko, R., 2002. The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis. Dev. Biol. 242, 376–387. Barth, A.D., Oko, R., 1989. Normal bovine spermatogenesis and sperm maturation. In: Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press, Ames, pp. 19–88. Cingolani, G., Petosa, C., Weis, K., Muller, C.W., 1999. Structure of importinbeta bound to the IBB domain of importin-alpha. Nature 399, 221–229. Clermont, Y., Oko, R., Hermo, L., 1993. Cell and molecular biology of the testis. In: Desjardins, C., Ewing, L. (Eds.), Cell Biology of
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