Skip to main content
PMC home page
Reproductive Biology and Endocrinology : RB&E logoLink to Reproductive Biology and Endocrinology : RB&E
. 2003 Feb 11;1:20. doi: 10.1186/1477-7827-1-20

Proposed mechanism for sperm chromatin condensation/decondensation in the male rat

John C Chapman 1, Sandra D Michael 1,✉
PMCID: PMC151792  PMID: 12646056

Abstract

Condensation of sperm chromatin occurs after spermatozoa have left the caput epididymis and are in transit to the cauda epididymis, during which time large numbers of disulfide bonds are formed. The formation of these disulfide bonds requires the repeated oxidation of the cofactor, NAD(P)H. To date, the means by which this oxidation is achieved has yet to be elucidated. Spermatozoa lose the bulk of their cytoplasm prior to leaving the testis; and, as a result, any shuttle systems for removing and transferring reducing equivalents into the mitochondria are unlikely to be operational. In an apparent preparation for the loss of cytoplasm, however, the following events occur during spermatogenesis. First, androgen-binding protein (ABP) is produced by the Sertoli cells of the testis; second, high affinity binding sites for ABP are inserted into the membrane surrounding the nucleus; and third, a nuclear location is acquired for the enzyme, 3α-hydroxysteroid dehydrogenase (3α-HSD).

We propose that after the loss of cytoplasm, the nuclear region of spermatozoa is directly accessible to constituents contained in the lumen of the caput epididymis. As a consequence, luminal ABP attaches itself to the nuclear membrane via its binding sites, and is internalized. After internalization, ABP exerts its principle function, which is to bind to luminal 5α-dihydrotestosterone (5α-DHT), thereby ensuring its availability to the enzyme, 3α-HSD. In the conversion of 5α-DHT to 3α-androstanediol (3α-Diol), NAD(P)H is oxidized. Spermatozoa that reach the cauda epididymis have fully condensed chromatin. In addition, the nuclear region retains appreciable amounts of 5α-DHT and 3α-Diol, both bound to ABP. During fertilization, the bound 3α-Diol is converted back to 5α-DHT, reducing equivalents are transferred to NAD(P)+, and disulfide bonds are broken.

IVF clinics report that spermatozoa with incompletely condensed chromatin have a low percentage of fertilization. If our proposed mechanism for chromatin condensation/decondensation is borne out by further research, IVF clinics might consider preincubating spermatozoa with 5α-DHT in order to increase the efficiency of fertilization.

Introduction

In eutherian mammals, the condensation of sperm chromatin has two main phases. The first phase, which occurs in the testis, involves the substitution of somatic histones by testis-specific protamines [1,2]. Protamines are small, only half the size of the core histones they replace, and are extremely basic. Between 55% and 70% of the amino acids are arginine. Sperm protamines also contain numerous cysteine residues, which are used to generate disulfide cross-links between adjacent protamine molecules during chromatin condensation. Bull sperm protamine contains 47 amino acids, with 24 arginine and 6 cysteine residues [3]; and rat sperm protamine consists of 50 amino acids, with 32 arginine and 5 cysteine residues [4]. Both protamine molecules are of sufficient length to fill one turn of DNA, with adjacent protamines locked in place around DNA by multiple disulfide bridges [3].

The formation of large numbers of disulfide cross-links between protamine molecules describes what occurs in the second main phase of chromatin condensation. These cross-links are formed after the spermatozoa have exited the caput epididymis and are in route to the cauda epididymis [5-9]. In the rat, the head region of spermatozoa contains approximately 6.9 nMoles of sulfhydryl groups (SH) + disulfides (SS) per million sperm, a figure which remains constant throughout spermatogenesis [10]. Spermatozoa that are isolated from the caput epididymis contain 84% of total SH + SS groups in the head region as thiols; whereas, sperm heads from the cauda epididymis contain only 14% of total SH + SS groups as thiols. This difference indicates that during transit between the two epididymides, almost 1.5 billion disulfide bonds are formed per individual sperm. Therefore, it is not surprising that after chromatin condensation, sperm are highly resistant to a variety of agents such as strong acids, proteases, DNAse, and detergents [11]. The overall effect of chromatin condensation is a transient inactivation of the male genome [12].

Chromatin condensation is directly related to the capacity of sperm to fertilize the ovum. For example, spermatozoa from both the caput epididymis and the proximal corpus epididymis lack the ability to fertilize; whereas, spermatozoa from the distal corpus epididymis and the cauda epididymis have this ability [8,13,14]. Human spermatozoa in which the chromatin is not completely condensed are also reported to have a low percentage of fertilization [15]. In a recent study, human sperm that were incompletely condensed failed to fertilize, even after their injection directly into the ovum [16]. Incomplete chromatin condensation is independent of other causes of infertility, such as abnormalities in sperm morphology (teratozoospermia), low sperm count (oligospermia), or poor sperm motility (asthenozoospermia) [17]. It has been suggested that incompletely condensed sperm constitute a significant factor in the assessment of male fertility [17].

In contrast to spermatogenesis, the process of fertilization requires that disulfide bonds between protamine molecules be broken. This occurs before chromatin decondensation, pronucleus formation, and DNA synthesis [18-21]. It has been proposed that glutathione, which is present in the egg cytoplasm, provides the reducing equivalents for the reduction of the disulfide bonds [18]. Under in vitro conditions, heparin-reduced glutathione does cause sperm decondensation [22]. The possibility that mitochondria, located in the middle piece of the spermatozoan, might be involved in decondensation via a lactate/pyruvate shuttle system [23] has also been considered. However, when spermatozoa were treated with cyanide, there was no effect on chromatin decondensation [24]. It has also been suggested that chromatin decondensation is the result of a trypsin-like, acrosomal protease that causes a proteolytic degradation of sperm protamine [25].

There is no question that the oxidation and reduction of sulfhydryl groups is critical to sperm chromatin condensation/decondensation. However, very little is known about the processes, or whether each utilizes the same mechanisms. The usual recipient for reducing equivalents is NAD(P)+, which is reduced to NAD(P)H. Unless the nuclear region contains an unlimited supply of NAD(P)+, it is critical that NAD(P)H transfer its reducing equivalents to some other molecule. During the early stages of spermatogenesis, reducing equivalents can be transferred from the cytoplasm into the mitochondria via shuttle systems [26,27]. However, spermatozoa lack cytoplasm, and their mitochondria are located in the middle piece [28]. Without cytoplasm it is unlikely that spermatozoa can transfer reducing equivalents from the head region to the middle piece. It has been reported that spermatozoa contain a membrane-bound NADPH oxidase for the transfer of reducing equivalents [29,30]. However, it was also reported that this NAD(P)H oxidase activity is insignificant [31], which limits the likelihood of it being a substitute for the shuttle systems. This leaves the question unanswered of how reducing equivalents are transferred in spermatozoa. The unique structure of spermatozoa, relative to that of a typical cell, suggests that their pathway for oxidizing NAD(P)H is unique as well. We previously reported that the head region of sonication-resistant spermatids converts endogenous 5α-dihydrotestosterone (5α-DHT) to 3α-androstanediol (3α-Diol) [32], a reaction in which NAD(P)H transfers its reducing equivalents to 5α-DHT. In our study, no cofactor was added to the incubation, indicating that endogenous NAD(P)H was the source of the reducing equivalents. Evidence that spermatozoa are also capable of this transfer is indicated by the report that bovine spermatozoa convert 3H-DHT to 3H-3α-Diol without added cofactor [33]. The remainder of this paper will use published data to develop and describe the putative role of the principle constituents in this unique pathway for reducing equivalents.

Hypothesis

Proposed mechanism for sperm chromatin condensation/decondensation

The first constituent in the proposed mechanism is androgen binding-protein (ABP). ABP is one of the major secretory products of the Sertoli cells of the mammalian testis [34-37]. In the rat, Sertoli cells secrete 20% of their ABP across the basal membranes into the interstitial compartment and 80% into the lumen of the seminiferous tubules [38,39]. ABP is then transported via the rete testis and efferent ductales into the caput and caudal epididymides [40-44]. During transit, the levels of ABP increase, and then fall in the cauda epididymis [45]. For example, in seminiferous tubule fluid the level of ABP is 40 nM; in rete testis fluid it is 60 nM; in the lumen of the caput epididymis it is 265 nM; and, in the lumen of the cauda epididymis it is 65 nM. Biologically active ABP can be detected in the serum of the male rat at 15 days of age [46,47]. However, after 40 days of age, the serum of the adult male rat contains less than 0.2% of the ABP measured in testis and caput epididymis [45]. In vitro and in vivo studies have demonstrated that the synthesis and secretion of ABP is regulated by androgens and FSH [48-50]. Testicular ABP has been found in all species that have been examined. The best characterized are rat, rabbit, and human ABP/SHBG (steroid hormone binding globulin) [51]. Despite testicular ABP being produced by the Sertoli cells and plasma SHBG originating from hepatocytes [52], it is now known that ABP and SHBG are encoded by the same gene and share a number of identical amino acid sequences [53-55].

It is generally accepted that one mole of ABP binds one mole of steroid. Although there is some variation in steroid specificity, ABP from most species binds DHT, T, estradiol-17-β (E2), and 5α-diol with high affinity. The dissociation constant (Kd) of ABP for DHT is between 1.6 × 10-9 M and 0.8 × 10-9 M. Testosterone, E2, and 5α-diol bind with lower affinities, but generally within one order of magnitude of that of DHT [42,56-59]. ABP does not appear to have any binding affinity for either androstenedione or progesterone.

Despite the extensive amount of data that have accumulated on the ABP molecule per se, very little definitive information on its role in male reproduction has been obtained. The underlying problem contributing to the lack of knowledge is that there are no known natural mutants in humans or animals where ABP is totally absent. This suggests that ABP is extremely important for mammalian development (i.e., mutants are lethal), or conversely, it is of little or no importance. The latter would appear unlikely considering the homology of sequence and activity of mammalian ABP, irrespective of species [51,59]. Early studies indicated that there is a correlation between decreased levels of ABP and infertility in: the pregnenolone-treated rat [60], the restricted rat [61], and hamsters exposed to altered photoperiods [62]. A series of papers by Huang et al. [48,63,64] indicated that the ability of spermatogenesis to produce viable sperm is closely related to ABP levels.

Sertoli cell cultures enriched with germ cells (spermatogonia, primary spermatocytes) undergo a doubling in the secretion of ABP [65,66]. The increased secretion of ABP requires FSH stimulation and the direct contact of the Sertoli cells with the spermatocytes. In 1984, Steinberger's group [67] reported that rat spermatocytes contain specific binding sites for ABP. Pelliniemi et al. [68], using antiABP antibodies, reported the presence of positive granules within the cytoplasm of spermatocytes and spermatids. Further use of the technique of immunocytochemistry has shown that the intensity of immunoreactive ABP staining and its intracellular localization in rat testis are dependent on the stage of the spermatogenic cycle [69].

Using purified ABP complexed to 3H-testosterone, Gerard et al. [70] demonstrated the endocytosis of ABP/SHBG by coated vesicles in monkey germ cells. The ligand-ABP complex was taken up by spermatogonia, spermatocytes, and early spermatids. This group reported that late spermatids and sperm did not internalize the ABP/SHBG. In a second study, using transmission electron microscopy and autoradiography, Gerard et al. [71] examined the internalization of ABP by rat germ cells and found that ABP was internalized by spermatocytes, round spermatids, and elongated spermatids. It was also noted that the intracellular site of ABP accumulation changed as the sperm matured. For example, labeling was most intense in nuclei having the less condensed form of chromatin. A nuclear location for ABP during the early stages of spermatogenesis suggests that it might play a role in transcription. Reports that stage XI elongated spermatids contain the androgen receptor [72], and synthesize mRNA [28], tend to bear this out.

ABP has also been shown to be associated with sperm during the later stages of spermatogenesis. For example, in an investigation of the endocytosis of ABP by sperm, Felden et al. [73], and Gerard [74] reported that rat germ cells each have 12,000 to 13,000 binding sites for ABP. The binding sites are a single class with a dissociation constant (Kd) for ABP of 0.78 nM [74]. It was proposed that the endocytosis of ABP is receptor mediated and related to the ABP binding activity previously identified on germ cell plasma membranes [71]. This suggests that ABP may also function in spermatogenesis as a steroid trans-membrane carrier. While this has yet to be demonstrated, it has been reported that tubules of the caput epididymis accumulate 3H-testosterone more efficiently from the luminal surface in the presence of ABP [75]. Others have suggested that the function of ABP is to establish and maintain the high androgen levels required for sperm maturation in the epididymides [45]. High levels of immunoreactive ABP have been found in the lumen of the initial segment of the caput epididymis, where the protein is seen to be coating both the spermatozoa and the brush border of the principal cells. Considerable staining is also observed in the epithelial cells, with the heaviest staining in the apical portion of the cells. The heavy staining of spermatozoa and epithelial cells in this region suggests a considerable degree of endocytosis of luminal ABP. Very little immunoreactive ABP is detected in the epithelial cells of the distal caput, corpus, and cauda epididymides [68].

The second constituent in the proposed pathway for transferring reducing equivalents is the enzyme, 3α-hydroxysteroid dehydrogenase (3α-HSD). This enzyme is believed to exist to limit the levels of DHT and to aid in the metabolic clearance of potent androgens. The clearance pathway for androgens is understood to be: testosterone → 5α-DHT → 3α-Diol glucuronide [76,77]. In humans, there are 4 isoforms of 3α-HSD, all within the aldo-keto reductase (AKR) superfamily [77]. The isoforms are NAD(P)(H)-dependent and convert 5α-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one [5α-DHT]) to yield 5α-androstane-3α, 17β-diol; [3α-Diol]). At present, different isoforms of 3α-HSD have been found in liver, prostate, mammary glands, uterus, brain, and skin [78,79]. In the oxidation direction, only AKR1C2, which is found in the brain, is able to convert 3α-Diol to 5α-DHT.

In the rat, there is a single form of 3α-HSD [80]. The reported locations for 3α-HSD in the male rat are liver, scrotal skin, muscle, prostate, epididymis, and sonication-resistant spermatids [32,80-82]. The epididymis of the adult male rat converts 3α-diol to 5α-DHT [82], indicating that the single form of 3α-HSD is reversible.

The head region of sonication-resistant rat spermatids converts endogenous bound 5α-DHT to 3α-Diol in the absence of added NAD(P)H cofactor [32]. In addition, bovine epididymal spermatozoa and ejaculated bovine sperm convert 3H-DHT to 3H-Diols, also without added cofactor [33]. This suggests that spermatozoa contain the enzyme, 3α-HSD, and that it utilizes endogenous NAD(P)H. It was also reported that 3H-DHT and 3H-Diols become bound to bovine spermatozoa during incubation [33].

The large numbers of disulfide bonds that are formed during chromatin condensation necessitate the repeated oxidation of NAD(P)H. We believe oxidation occurs via the transfer of reducing equivalents to 5α-DHT. The overall process requires the concerted efforts of the enzyme 3α-HSD, and ABP. Unlike 5α reductase, an enzyme found primarily in spermatocytes [83,84], 3α-HSD is found in spermatids [32], as well as in spermatozoa [33]. With 3α-HSD localized in the nuclear region of the sperm [32], the only limitation on the oxidation of NAD(P)H is the availability of 5α-DHT, which is the responsibility of ABP. The nuclear region of each spermatozoan is enclosed by a membrane that has 12,000 to 13,000 high affinity binding sites for ABP [73,74]. These ABP binding sites come into play after the loss of cytoplasm and the spermatozoa have entered the caput epididymis. Here, immunoreactive ABP can be seen covering the spermatozoa [69]. The binding and internalization of ABP, as well as the subsequent delivery of 5α-DHT, is facilitated by the extremely high levels of both ABP and 5α-DHT, which in the caput epididymis are 265 nM and 200 nM, respectively [45]. The observation that ABP is not found in epithelial cells of the distal caput, corpus, and cauda epididymides [68], suggests that the internalization of ABP is limited to the proximal caput epididymis.

We have examined the head region of spermatozoa, isolated from the cauda epididymis for the presence of bound androgens, and detected 3α-Diol (1344 pg/mg DNA) and 5α-DHT (385 pg/mg DNA) [85]. Both androgens were tightly bound to the head region and remained there even when spermatozoa were isolated from the cauda epididymis of male rats that had been castrated three days previously. The obvious tenacity of the head region for these two androgens is very likely due to ABP. Evidence for the continued presence of ABP in mature spermatozoa can be provided by calculating the theoretical levels of bound androgen, and comparing this figure with the actual levels of bound androgen. Since the nuclear region of each spermatozoan contains 3.5 pg DNA [85], and has between 12,000 to 13,000 binding sites for ABP [73,74], the theoretical level of bound androgen would be between 1650 pg/mg DNA and 1790 pg/mg DNA. The measured levels of 3α-Diol and 5α-DHT were 1344 pg/mg DNA and 385 pg/mg DNA, respectively, or a total of 1729 pg/mg DNA [85].

In the formation of disulfide bonds, NAD(P)H has to be repeatedly oxidized. During fertilization and the dissolution of disulfide bonds, the reverse occurs, and NAD(P)+ has to be repeatedly reduced. If chromatin decondensation utilizes the mechanism of chromatin condensation, but in reverse, then this will occur through the transfer of reducing equivalents from 3α-Diol to NAD(P)+. We have found that the 3α-Diol bound to the head region is consistently 3 fold that of bound 5α-DHT [85], indicating that 3α-HSD is functioning as a reductase. For the enzyme to operate in the reverse direction, it is likely that activation by an external source is required. This activation could be in the form of a female sex hormone, such as estrogen or progesterone. Progesterone is known to act on the sperm to induce capacitation [86]. It could also cause 3α-HSD to function as an oxidase. There are approximately 10,000 molecules of 3α-Diol per spermatozoan [85]. This number would produce less than 1% of the reducing equivalents needed to break all disulfide bounds. Still, enough bonds could be broken to allow reduced glutathione, contained in the cytoplasm of the ovum [18], to penetrate the chromatin and affect the dissolution of the remainder of the disulfide bonds.

It has been suggested that glutathione is an intermediary in sperm condensation [29]. If this is indeed the case, reducing equivalents still need to be transferred, for which our proposed mechanism is applicable.

Testing the proposed mechanism for sperm chromatin condensation

The literature contains a number of reports that tentatively support our proposed mechanism. For example, a reduction in androgen levels in the lumen of the caput and cauda epididymides is correlated with decreased numbers of disulfide bonds in spermatozoa [87,88]. In addition, there is a significant correlation between ABP levels and sperm fertilizing ability in the pregnenolone-injected rat [60] and the restricted (Hre) rat [89]. In both experimental animals, decreased levels of ABP caused a defect in sperm quality, but had no affect on sperm quantity. While these studies provide supportive evidence, the best way to validate the proposed mechanism is to demonstrate that the formation of disulfide bonds in caput epididymal spermatozoa is completely dependent upon the conversion of 5α-DHT to 3α-Diol.

Significance of the proposed mechanism for sperm chromatin condensation

Knowledge of the actual mechanism of chromatin condensation/decondensation is important to the field of reproductive endocrinology. For example, if 5α-DHT is found to be the recipient of reducing equivalents during chromatin condensation, IVF clinics might consider preincubating spermatozoa with 5α-DHT in order to increase the efficiency of fertilization.

Contributor Information

John C Chapman, Email: johnchapman1@juno.com.

Sandra D Michael, Email: smichael@binghamton.edu.

References

  1. Bellve AR, Anderson E, Hanley-Bowdoin L. Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei. Dev Biol. 1975;47:349–365. doi: 10.1016/0012-1606(75)90289-4. [DOI] [PubMed] [Google Scholar]
  2. Goldberg RB, Geremia R, Bruce WR. Histone synthesis and replacement during spermatogenesis in the mouse. Differentiation. 1977;7:167–180. doi: 10.1111/j.1432-0436.1977.tb01508.x. [DOI] [PubMed] [Google Scholar]
  3. Coelingh JP, Monfoort CH, Rozijn TH, Gevers Leuven JA, Schiphof R, Steyn-Parve EP, Braunitzer G, Schrank B, Ruhfus A. The complete amino acid sequence of the basic nuclear protein of bull spermatozoa. Biochem Biophys Acta. 1972;285:1–14. doi: 10.1016/0005-2795(72)90174-2. [DOI] [PubMed] [Google Scholar]
  4. Marushige Y, Marushige K. Transformation of sperm histone during formation and maturation of rat spermatozoa. J Biol Chem. 1975;250:39–45. [PubMed] [Google Scholar]
  5. Aravindan GR, Krishnamurthy H, Moudgal NR. Rat epididymal sperm exhibit on dithiothreitol treatment in vitro quantifiable differences in patterns of light scatter, uptake of 14C-iodoacetamide and binding of ethidium bromide to DNA. Indian J Exp Biol. 1995;33:899–910. [PubMed] [Google Scholar]
  6. Hingst O, Blottner S, Franz C. Chromatin condensation in cat spermatozoa during epididymal transit as studied by aniline blue and acridine orange staining. Andrologia. 1995;27:275–279. doi: 10.1111/j.1439-0272.1995.tb01105.x. [DOI] [PubMed] [Google Scholar]
  7. Haidl G, Badura B, Schill WB. Function of human epididymal spermatozoa. J Androl. 1994;15:23S–27S. [PubMed] [Google Scholar]
  8. Haidl G. Epididymal maturation of human spermatozoa. Fortschr Med. 1994;112:492–495. [PubMed] [Google Scholar]
  9. Golan R, Cooper TG, Oschry Y, Oberpenning F, Schulze H, Shochat L, Lewin LM. Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry. Hum Reprod. 1996;11:1457–1462. doi: 10.1093/oxfordjournals.humrep.a019419. [DOI] [PubMed] [Google Scholar]
  10. Shalgi R, Seligman J, Kosower NS. Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane. Biol Reprod. 1989;40:1037–1045. doi: 10.1095/biolreprod40.5.1037. [DOI] [PubMed] [Google Scholar]
  11. Mahi CA, Yanagimachi R. Induction of nuclear decondensation of mammalian sperm in vitro. J Reprod Fertil. 1975;44:293–296. doi: 10.1530/jrf.0.0440293. [DOI] [PubMed] [Google Scholar]
  12. Bedford JM, Calvin HI. The occurrence and possible functional significance of -S-S- crosslinks in sperm heads, with particular reference to eutherian mammals. J Exp Zool. 1974;188:137–158. doi: 10.1002/jez.1401880203. [DOI] [PubMed] [Google Scholar]
  13. Orgebin-Crist MC, Jahad N, Hoffman LH. The effects of testosterone, 5alpha-dihydrotestosterone, 3alpha-androstanediol, and 3beta-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture. Cell Tissue Res. 1976;167:515–525. doi: 10.1007/BF00215181. [DOI] [PubMed] [Google Scholar]
  14. Weissenberg R, Yossefi S, Oschry Y, Madgar I, Lewin LM. Investigation of epididymal sperm maturation in the golden hamster. Int J Androl. 1994;17:256–261. doi: 10.1111/j.1365-2605.1994.tb01251.x. [DOI] [PubMed] [Google Scholar]
  15. Hammadeh ME, Steiber M, Haidl G, Schmidt W. Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program. Andrologia. 1998;30:29–35. doi: 10.1111/j.1439-0272.1998.tb01379.x. [DOI] [PubMed] [Google Scholar]
  16. Rosenbusch BE. Frequency and patterns of premature sperm chromosome condensation in oocytes failing to fertilize after intracytoplasmic sperm injection. J Assist Reprod Genet. 2000;17:253–259. doi: 10.1023/A:1009454231659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hammadeh ME, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl. 2001;46:99–104. doi: 10.1080/01485010151093958. [DOI] [PubMed] [Google Scholar]
  18. Zirkin BR, Perreault SD, Naish SJ. Formation and function of the male pronucleus during mammalian fertilization. In: Schatten H, Schatten G, editor. The Molecular Biology of Fertilization. New York, Academic Press. Harcourt Brace Jovanovich; 1989. pp. 91–114. [Google Scholar]
  19. Perreault SD, Wolff RA, Zirkin BR. The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev Biol. 1984;101:160–167. doi: 10.1016/0012-1606(84)90126-x. [DOI] [PubMed] [Google Scholar]
  20. Miller MA, Masui Y. Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm-oocyte interactions in mice. Gamete Res. 1982;5:167–179. [Google Scholar]
  21. Longo FL. Regulation of pronuclear development. In: Jagiello G, Vogel HJ, editor. Bioregulators of Reproduction. New York, Academic Press; 1981. pp. 529–557. [Google Scholar]
  22. Reyes R, Sanchez-Vazquez ML, Merchant-Larios H, Rosado A, Delgado NM. Effect of heparin-reduced glutathione on hamster sperm DNA unpacking and nuclear swelling. Arch Androl. 1996;37:33–45. doi: 10.3109/01485019608988500. [DOI] [PubMed] [Google Scholar]
  23. Gallina-Fernando G, Gerez de Burgos NM, Burgos C, Coronel CE, Blanco A. The lactate/pyruvate shuttle in spermatozoa: operation in vitro. Arch Biochem Biophys. 1994;308:515–519. doi: 10.1006/abbi.1994.1072. [DOI] [PubMed] [Google Scholar]
  24. Ahmadi A, Ng-Soon C. Sperm head decondensation, pronuclear formation, cleavage and embryonic development following intracytoplasmic injection of mitochondria-damaged sperm in mammals. Zygote. 1997;5:247–253. doi: 10.1017/s0967199400003683. [DOI] [PubMed] [Google Scholar]
  25. Marushige Y, Marushige K. Enzymatic unpacking of bull sperm chromatin. Biochem Biophys Acta. 1975;403:180–191. doi: 10.1016/0005-2744(75)90020-0. [DOI] [PubMed] [Google Scholar]
  26. Burgos C, Coronel CE, de Burgos NM, Rovai LE, Blanco A. Studies in vitro on shuttle systems of mouse mitochondria. Biochem J. 1982;208:413–417. doi: 10.1042/bj2080413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Coronel CE, Gallina FG, Gerez de Burgos NM, Burgos C, Blanco A. Properties of the branched-chain 2-hydroxy acid/2-oxo acid shuttle in mouse spermatozoa. Biochem J. 1986;235:853–858. doi: 10.1042/bj2350853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Monesi V. Spermatogenesis and the spermatozoa. In: Austin CR, Short RV, editor. Reproduction in Mammals. I. Germ Cells and Fertilization. New York, Cambridge University Press; 1972. pp. 46–84. [Google Scholar]
  29. Aitken RJ, Vernet P. Maturation of redox regulatory mechanisms in the epididymis. J Reprod Fertil Suppl. 1998;53:109–118. [PubMed] [Google Scholar]
  30. Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod. 2001;65:1102–1113. doi: 10.1095/biolreprod65.4.1102. [DOI] [PubMed] [Google Scholar]
  31. Richer SC, Ford WC. A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Hum Reprod. 2001;7:237–244. doi: 10.1093/molehr/7.3.237. [DOI] [PubMed] [Google Scholar]
  32. Chapman JC, Freeh SM, Michael SD. Sonication-resistent rat spermatid heads convert endogenous 5 alpha dihyrotestosterone to 5 alpha androstanediol. Med Sci Res. 1993;21:903–904. [Google Scholar]
  33. Djoseland O, Hogo S, Nyberg K, Hastings CD, Attramadal A. Uptake and metabolism of androgens by bovine spermatozoa. Steroids. 1978;31:307–317. doi: 10.1016/0039-128X(78)90045-4. [DOI] [PubMed] [Google Scholar]
  34. Hagenas L, Ritzen EM, Plooen L, Hansson V, French FS, Nayfeh SN. Sertoli cell origin of testicular androgen-binding protein (ABP). Mol Cell Endocrinol. 1975;2:339–350. doi: 10.1016/0303-7207(75)90021-0. [DOI] [PubMed] [Google Scholar]
  35. Steinberger A, Heindel JJ, Lindsey JN, Elkington JSH, Sanborn BM, Steinberger E. Isolation and culture of FSH responsive Sertoli cells. Endocr Res Commun. 1975;2:261–272. doi: 10.3109/07435807509053853. [DOI] [PubMed] [Google Scholar]
  36. Louis BG, Fritz IB. Follicle-stimulating hormone and testosterone independently increase the production of androgen binding protein by Sertoli cells in culture. Endocrinology. 1979;104:454–461. doi: 10.1210/endo-104-2-454. [DOI] [PubMed] [Google Scholar]
  37. Schmidt WN, Taylor CA, Danzo BJ. The use of photoaffinity ligand to compare androgen-binding protein (ABP) present in rat Sertoli cell culture media with ABP present in epididymal cytosol. Endocrinology. 1981;108:786–794. doi: 10.1210/endo-108-3-786. [DOI] [PubMed] [Google Scholar]
  38. Gunsalus GL, Musto NA, Bardin CW. Bidirectional release of a Sertoli cell product, androgen binding protein, into the blood and seminiferous tubule. In: Steinberger A, Steinberger E, editor. Testicular development, structure, and function. New York, Raven Press; 1980. pp. 291–297. [Google Scholar]
  39. Gunsalus GL, Bardin CW. Sertoli-germ cell interactions as determinants of bi-directional secretion of androgen-binding protein. Ann NY Acad Sci. 1991;637:322–326. doi: 10.1111/j.1749-6632.1991.tb27319.x. [DOI] [PubMed] [Google Scholar]
  40. French FS, Ritzen EM. A high affinity androgen-binding protein (ABP) in rat testis: evidence for secretion into efferent duct fluid and absorption by epididymis. Endocrinology. 1973;93:88–95. doi: 10.1210/endo-93-1-88. [DOI] [PubMed] [Google Scholar]
  41. Danzo BJ, Eller BC, Orgebin-Crist MC. Studies on the site of origin of androgen binding protein present in epididymal cytosol from mature intact rabbits. Steroids. 1974;24:107–122. doi: 10.1016/0039-128X(74)90049-X. [DOI] [PubMed] [Google Scholar]
  42. Vernon RG, Kopec B, Fritz IB. Observations on the binding of androgens by rat testis seminiferous tubules and testis extracts. Mol Cell Endocrinol. 1974;1:167–187. doi: 10.1016/0303-7207(74)90028-8. [DOI] [Google Scholar]
  43. Hansson V, Ritzen EM, French FS, Nayfeh SN. Androgen transport and receptor mechanisms in testis and epididymis. In: Hamilton DW, Greep RO, editor. Handbook of Physiology. Sect. 7. Vol. 5. Washington, D.C., American Physiological Society; 1975. pp. 173–201. [Google Scholar]
  44. Fritz IB. Sites of action of androgens and follicle stimulating hormone on cells of the seminiferous tubule. In: Litwack G, editor. Biochemical Actions of Hormones. Vol. 5. New York, Academic Press; 1978. pp. 249–281. [Google Scholar]
  45. Turner TT, Jones CE, Howards SS, Ewing LL, Zegeye B, Gunsalus GL. On the androgen microenvironment of maturing spermatozoa. Endocrinology. 1984;115:1925–1932. doi: 10.1210/endo-115-5-1925. [DOI] [PubMed] [Google Scholar]
  46. Danzo BJ, Eller BC. The ontogeny of biologically active androgen-binding protein in rat plasma, testis, and epididymis. Endocrinology. 1985;117:1380–1388. doi: 10.1210/endo-117-4-1380. [DOI] [PubMed] [Google Scholar]
  47. Nazian SJ. Concentrations of free testosterone, total testosterone, and androgen binding protein in the peripheral serum of male rats during sexual maturation. J Androl. 1986;7:49–54. doi: 10.1002/j.1939-4640.1986.tb00867.x. [DOI] [PubMed] [Google Scholar]
  48. Huang HFS, Pogach LM, Nathan E, Giglio W, Seebode JJ. Synergistic effects of follicle-stimulating hormone and testosterone on the maintenance of spermiogenesis in hypophysectomized rats: relationship with the androgen-binding protein status. Endocrinology. 1991;128:3152–3161. doi: 10.1210/endo-128-6-3152. [DOI] [PubMed] [Google Scholar]
  49. Hansson V, Reusch E, Trygstad O, Torgersen O, Ritzen EM, French FS. FSH stimulation of testicular androgen binding protein. Nature. 1973;246:56–58. doi: 10.1038/newbio246056a0. [DOI] [PubMed] [Google Scholar]
  50. Ritzen EM, Hagenas L, Hansson V, French FS. In vitro synthesis of testicular androgen binding protein (ABP); stimulation by FSH and androgen. In: French FS, Hansson V, Ritzen EM, Nayfeh SN, editor. Hormonal Regulation of Spermatogenesis. New York, Plenum Press; 1975. pp. 353–366. [Google Scholar]
  51. Westphal U. II. Monographs on Endocrinology. Berlin, Springer-Verlag; 1986. Steroid-protein interactions. pp. 198–301. [PubMed] [Google Scholar]
  52. Khan MS, Knowles BB, Aden DP, Rosner W. Secretion of testosterone-estradiol-binding globulin by a human hepatoma-derived cell line. J Clin Endocrin Metab. 1981;53:448–449. doi: 10.1210/jcem-53-2-448. [DOI] [PubMed] [Google Scholar]
  53. Joseph DR, Hall SH, Conti M, French FS. The gene structure of rat androgen-binding protein: identification of potential regulatory deoxyribonucleic acid elements of a follicle-stimulating hormone-regulated protein. Molec Endocr. 1988;2:3–13. doi: 10.1210/mend-2-1-3. [DOI] [PubMed] [Google Scholar]
  54. Gershagen S, Lundwall A, Fernlund P. Characterization of the human sex hormone-binding globulin (SHBG) gene and demonstration of two transcripts in both liver and testis. Nucleic Acids Res. 1989;17:9245–9258. doi: 10.1093/nar/17.22.9245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sullivan PM, Petrusz P, Szpirer C, Joseph DR. Alternative processing of androgen-binding protein RNA transcripts in fetal rat liver. Identification of a transcript formed by trans splicing. J Biol Chem. 1991;266:143–154. [PubMed] [Google Scholar]
  56. Cunningham GR, Tindall DJ, Means AR. Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor. Steroids. 1979;33:261–276. doi: 10.1016/0039-128X(79)90003-5. [DOI] [PubMed] [Google Scholar]
  57. French FS, Ritzen EM. Androgen-binding protein in efferent duct fluid of rat testis. J Reprod Fert. 1973;32:479–483. doi: 10.1530/jrf.0.0320479. [DOI] [PubMed] [Google Scholar]
  58. Ritzen EM, French FS, Weddington SC, Nayfeh SN, Hansson V. Steroid binding in polyacrylamide gels. Quantitation at steady state conditions. J Biol Chem. 1974;249:6597–6604. [PubMed] [Google Scholar]
  59. Danzo BJ. The effects of a gonadotropin-releasing hormone antagonist on androgen-binding protein distribution and other parameters in the adult male rat. Endocrinology. 1995;136:4004–4011. doi: 10.1210/endo.136.9.7649109. [DOI] [PubMed] [Google Scholar]
  60. Anthony CT, Danzo BJ, Orgebin-Crist M-C. Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the hypophysectomized, pregnenolone-injected rat. Endocrinology. 1984;114:1419–1425. doi: 10.1210/endo-114-4-1419. [DOI] [PubMed] [Google Scholar]
  61. Anthony CT, Danzo BJ, Orgebin-Crist MC. Investigations on the relationship between sperm fertilizing ability and androgen binding protein in the restricted rat. Endocrinology. 1984;114:1413–1418. doi: 10.1210/endo-114-4-1413. [DOI] [PubMed] [Google Scholar]
  62. Holland MK, Rogers BJ, Orgebin-Crist MC, Danzo BJ. Effects of photoperiod on androgen-binding protein and sperm fertilizing ability in the hamster. J Reprod Fertil. 1987;81:99–112. doi: 10.1530/jrf.0.0810099. [DOI] [PubMed] [Google Scholar]
  63. Huang HFS, Pogach L, Giglio W, Nathan E, Seebode J. GnRH-A induced arrest of spermiogenesis in rats is associated with altered androgen binding protein distribution in the testis and epididymis. J Androl. 1992;13:153–159. [PubMed] [Google Scholar]
  64. Pogach L, Giglio W, Nathan E, Huang HFS. Maintenance of spermiogenesis by exogenous testosterone in rats treated with a GnRH antagonist: relationship with androgen-binding protein status. J Reprod Fert. 1993;98:415–422. doi: 10.1530/jrf.0.0980415. [DOI] [PubMed] [Google Scholar]
  65. Galdieri M, Monaco L, Stefanini M. Secretion of androgen binding protein by Sertoli cells is influenced by contact with germ cells. J Androl. 1984;5:409–415. doi: 10.1002/j.1939-4640.1984.tb00806.x. [DOI] [PubMed] [Google Scholar]
  66. Janecki AJ, Jakubowiak A, Steinberger A. Effect of germ cells on vectorial secretion of androgen-binding protein and transferrin by immature rat Sertoli cells in vitro. J Androl. 1988;9:126–132. doi: 10.1002/j.1939-4640.1988.tb01024.x. [DOI] [PubMed] [Google Scholar]
  67. Steinberger A, Dighe RR, Diaz J. Testicular peptides and their endocrine and paracrine functions. Arch Biol Med Exp. 1984;17:267–271. [PubMed] [Google Scholar]
  68. Pelliniemi LJ, Dym M, Gunsalus GL, Musto NA, Bardin CW, Fawcett DW. Immunocytochemical localization of androgen-binding protein in the male rat reproductive tract. Endocrinology. 1981;108:925–931. doi: 10.1210/endo-108-3-925. [DOI] [PubMed] [Google Scholar]
  69. Attramadal A, Bardin CW, Gunsalus GL, Musto NA, Hansson V. Immunocytochemical localization of androgen binding protein in rat sertoli and epididymal cells. Biol Reprod. 1981;25:983–988. doi: 10.1095/biolreprod25.5.983. [DOI] [PubMed] [Google Scholar]
  70. Gerard A, En Nya A, Egloff M, Domingo M, Degrelle H, Gerard H. Endocytosis of human sex steroid-binding protein in monkey germ cells. Ann NY Acad Sci. 1991;637:258–276. doi: 10.1111/j.1749-6632.1991.tb27314.x. [DOI] [PubMed] [Google Scholar]
  71. Gerard H, Gerard A, En Nya A, Felden F, Gueant JL. Spermatogenic cells do internalize Sertoli cell androgen-binding protein: a transmission electron microscopy autoradiographic study in the rat. Endocrinology. 1994;134:1515–1527. doi: 10.1210/endo.134.3.8119194. [DOI] [PubMed] [Google Scholar]
  72. Vornberger W, Prins G, Musto NA, Suarez-Quian CA. Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology. 1994;134:2307–2316. doi: 10.1210/endo.134.5.8156934. [DOI] [PubMed] [Google Scholar]
  73. Felden F, Gueant JL, En Nya A, Gerard A, Fremont S, Nicolas JP, Gerard H. Photoaffinity labelled rat androgen-binding protein and sex hormone steroid-binding protein bind specifically to rat germ cells. J Mol Endocrinol. 1992;9:39–46. doi: 10.1677/jme.0.0090039. [DOI] [PubMed] [Google Scholar]
  74. Gerard A. Endocytosis of androgen-binding protein (ABP) by spermatogenic cells. J Steroid Biochem Mol Biol. 1995;53:533–542. doi: 10.1016/0960-0760(95)00109-D. [DOI] [PubMed] [Google Scholar]
  75. Porto CS, Lazari MFM, Abreu LC, Bardin CW, Gunsalus GL. Receptors for androgen-binding proteins: internalization and intracellular signaling. J Steroid Biochem Mol Biol. 1995;53:561–565. doi: 10.1016/0960-0760(95)00111-C. [DOI] [PubMed] [Google Scholar]
  76. Moghissi E, Ablan F, Horton R. Origin of plasma androstanediol glucuronide in men. J Clin Endocrinol Metab. 1984;59:417–421. doi: 10.1210/jcem-59-3-417. [DOI] [PubMed] [Google Scholar]
  77. Penning TM, Burczynski ME, Jez JM, Hung CF, Lin HK, Ma H, Moore M, Palackal N, Ratnam K. Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem J. 2000;351:67–77. doi: 10.1042/0264-6021:3510067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Duffy DM, Legro RS, Chang L, Stanczyk FZ, Lobo RA. Metabolism of dihydrotestosterone to 5 alpha-androstane-3 alpha, 17 beta-diol glucuronide is greater in the peripheral compartment than in the splanchnic compartment. Fertil Steril. 1995;64:736–739. doi: 10.1016/s0015-0282(16)57848-0. [DOI] [PubMed] [Google Scholar]
  79. Rittmaster RS, Zwicker H, Thompson DL, Konak G, Norman RW. Androstanediol glucuronide production in human liver, prostate, and skin. Evidence for the importance of the liver in 5 alpha-reduced androgen metabolism. J Clin Endocrinol Metab. 1993;76:977–982. doi: 10.1210/jcem.76.4.8473413. [DOI] [PubMed] [Google Scholar]
  80. Schlegel BP, Jez JM, Penning TM. Mutagenesis of 3 alpha-hydroxysteroid dehydrogenase reveals a "push-pull" mechanism for proton transfer in aldo-keto reductases. Biochemistry. 1998;37:3538–3548. doi: 10.1021/bi9723055. [DOI] [PubMed] [Google Scholar]
  81. Pasupuleta V, Horton R. Metabolism of 5 alpha reduced androgens by various tissues of the male rat. J Androl. 1990;11:161–167. [PubMed] [Google Scholar]
  82. Hastings CD, Djoseland O. Androgen metabolism by rat epididymis. Metabolic conversion of 3H 5 alpha-androstane-3 alpha, 17 beta-diol, in vitro. Steroids. 1977;30:531–539. doi: 10.1016/0039-128X(77)90098-8. [DOI] [PubMed] [Google Scholar]
  83. Dorrington JH, Fritz IB. Cellular localization of 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase in the seminiferous tubule of the rat testis. Endocrinology. 1975;96:879–889. doi: 10.1210/endo-96-4-879. [DOI] [PubMed] [Google Scholar]
  84. Dorrington JH, Fritz IB. Metabolism of testosterone by preparations from the rat testis. Biochem Biophys Res Commun. 1973;54:1425–1431. doi: 10.1016/0006-291x(73)91145-5. [DOI] [PubMed] [Google Scholar]
  85. Frankel AI, Chapman JC, Wright WW. The equivocal presence of nuclear androgen binding proteins in mammalian spermatids and spermatozoa. J Steroid Biochem. 1989;33:71–79. doi: 10.1016/0022-4731(89)90360-9. [DOI] [PubMed] [Google Scholar]
  86. de Lamirande E, Harakat A, Gagnon C. Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion. J Androl. 1998;19:215–225. [PubMed] [Google Scholar]
  87. Seligman J, Kosower NS, Shalgi R. Effects of castration on thiol status in rat spermatozoa and epididymal fluid. Mol Reprod Dev. 1997;47:295–301. doi: 10.1002/(SICI)1098-2795(199707)47:3<295::AID-MRD8>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  88. Huang HF, Nieschlag E. Alteration of free sulphydryl content of rat sperm heads by suppression of intratesticular testosterone. J Reprod Fertil. 1984;70:31–38. doi: 10.1530/jrf.0.0700031. [DOI] [PubMed] [Google Scholar]
  89. Musto NA, Bardin CW. Decreased levels of androgen binding protein in the reproductive tract of the restricted (Hre) rat. Steroids. 1976;28:1–11. doi: 10.1016/0039-128X(76)90120-3. [DOI] [PubMed] [Google Scholar]

Articles from Reproductive biology and endocrinology : RB&E are provided here courtesy of BMC

RESOURCES