C. H. Yeung

Sperm Volume Regulation: Maturational Changes in Fertile and Infertile Transgenic Mice and Association with Kinematics and Tail Angulation

Abstract Laser light scatter analyzed by flow cytometry was used to monitor the volume of viable maturing murine spermatozoa. Upon release, dispersion, and dilution, epididymal sperm from fertile heterozygous c-ros knockout mice were smallest in the cauda region and largest in the corpus region. Cauda sperm from both infertile homozygous c-ros knockout and GPX5-Tag2 transgenic mice were abnormally large. When incubated, corpus and cauda sperm from normal mice became slightly enlarged and later returned to a smaller size. This suggests an immediate swelling due to high intracellular osmolality, which triggers a regulatory volume decrease (RVD) that results in a net volume reduction. Normal caput sperm increased in size continuously and became larger than the more mature sperm, indicating a lack of RVD. The ion-channel blocker quinine induced dose-dependent size increases in normal cauda sperm but not in caput sperm. Dose-dependent quinine action on mature sperm also included induction of tail angulation, and suppression of straight-line velocity and linearity. The kinematic effects were more sensitive, with a quicker onset, but they diminished with time in contrast to tail angulation, which intensified. These results suggest that kinematic changes are an early phenomenon of swelling, which gradually accumulates at the cytoplasmic droplet to cause flagellar angulation. Disruption of the epididymal maturation of sperm volume regulation capacity would hinder the transport of sperm in the female tract, and may thereby explain infertility under certain conditions, but may also provide a novel approach to male contraception.

Aquaporin Isoforms Involved in Physiological Volume Regulation of Murine Spermatozoa

Abstract Murine epididymal spermatozoa were dispersed in a medium of native osmolality and then transferred to a hypo-osmotic medium to mimic the physiological osmotic challenge, as encountered upon ejaculation into the female tract. The addition of quinine to block sperm K+-channels for volume regulation resulted in a size increase of viable cells. Preincubation in 0.1 mM HgCl2, a standard aquaporin inhibitor, prevented such cell swelling. Addition of the K+-ionophore valinomycin to quinine-swollen sperm reversed the swelling, but not after pretreatment of the swollen sperm by HgCl2. Aqp7, Aqp8, and Aqp9 mRNAs were identified in spermatozoa by RT-PCR, and the entire open reading frames were sequenced and compared with the GenBank database. Western blotting demonstrated specific protein signals for sperm AQP7 and AQP8 expression but probably not AQP9. The role of Hg2+-insensitive AQP7, if any, in sperm volume regulation was studied in transgenic mice. Spermatozoa from Aqp7−/− mice were the same size as wild-type sperm in basal conditions. Quinine-swollen volume, swelling reversal by valinomycin, and inhibition by Hg2+ were also similar, indicating efficient water transport in the absence of AQP7. However, both water influx and efflux occurred faster in Aqp7−/− sperm than wild-type. This faster water movement in the knockout mouse spermatozoa was explainable by an upregulation of Aqp8 expression as revealed by quantitative PCR. Therefore, the Hg2+-sensitive AQP8, which was localized in elongated spermatids and spermatozoa, is a likely candidate for a water channel responsible for physiological sperm volume regulation crucial to in vivo fertilization.

Aquaporins in the human testis and spermatozoa -identification, involvement in sperm volume regulation and clinical relevance

Despite the high water-permeability of human spermatozoa, little is known about the identity and the role of aquaporins (AQP) in them or germ cells. Using ejaculates from donors, sperm AQPs were identified by western blotting followed by the analysis of mRNA with RT-PCR. Protein expression in the testis and spermatozoa was localized by immunocytochemistry. Inhibitors were used to investigate the involvement of aquaporins in water transport when ejaculated spermatozoa were swollen in medium mimicking uterine hypo-osmolality by quinine that blocks volume regulation. Sperm AQP7 and AQP8 in 39 infertile patients and 11 healthy donors were quantified by flow cytometry. AQP1 was absent from spermatozoa. Sperm and testicular AQP7-9 had nucleotide sequences identical to those of somatic cells but AQP8 mRNA also showed shorter variants. AQP7 was expressed abundantly by round and elongated spermatids and ejaculated spermatozoa, AQP8 by all germ cells and spermatozoa, and AQP9 rarely by spermatocytes or Sertoli cells. Protein bands showed specificity by western blotting for AQP7 and AQP8 but not AQP9. The absence of sperm AQP9 was further suggested by the ineffectiveness of its inhibitor phloretin in blocking quinine-induced swelling, but HgCl(2,) which inhibits AQP8, was effective. Sperm AQP7 expression was correlated with progressive motility and was lower in patients than in donors. Sperm AQP8 expression was inversely correlated with the extent of sperm coiling, which is a swelling phenomenon, but showed no difference between patients and donors. In conclusion, AQP7 and AQP8 were identified in human spermatozoa and could play a role in glycerol metabolism and water transport respectively.

Acquisition and Development of Sperm Motility Upon Maturation in the Epididymis

Although mature sperm in the cauda epididymidis are quiescent in their native fluid (except in the rabbit: Turner and Reich, 1985), they exhibit vigorous forward motility when diluted in culture medium. This motility is crucial for transport in the female tract (see Yanagimachi, 1994) and for penetration of the cumulus oophorus and zona pellucida (see Bedford, 1998). In contrast, testicular sperm are mostly immotile, except in rabbits (Perez-Sanchez et al., 1996). The acquisition of the potential for motility and development of the swimming pattern are the most obvious maturational changes of sperm in the epididymis. The application of CASA (computer-assisted sperm analysis) enables relatively objective and quantitative descriptions of such changes in terms of velocities and other kinematic parameters (see Perreault, this volume). These kinematic parameters can serve as sensitive indicators of sperm maturation in the study of epididymal regulatory factors, and the profile of change can also form the basis of reference for studying the development of other functional capacities of maturing sperm. Motility development involves first the acquisition of the potential for flagellation, and then the coordination and modulation of the flagellar waveform resulting in the characteristic mature swimming pattern. This chapter reviews the current understanding of the initiation of flagellation and the regulation of the flagellar waveform, as revealed by the ultrastructural basis and the molecular mechanisms for microtubule sliding and its translation into axonemal bending and oscillation. The important role of the outer dense fibres with their maturational changes in the regulation of motility pattern, and the involvement of intracellular factors as well as epididymal secretions are discussed.

Physiology of Sperm Maturation and Fertilization

The primary sex organ, the testis, concerned with the manufacture of androgens and spermatozoa, is in continuity with its extra-testicular pathways (the ductuli efferentia, epididymis and ductus deferens), that transmit sperm to the urethra. Also discharging their secretions into the urethra are the more distal secondary sexual organs, the prostate and seminal vesicles, and the periurethral glands (of Littre) and bulbourethral glands (of Cowper) (Fig. 4.1).

Coiled sperm from infertile patients: characteristics, associated factors and biological implication

BACKGROUNDnThere is no systematic study on coiled sperm in semen, although they are commonly observed. This work characterizes coiled sperm in infertile men to understand the clinical implications and investigate the possible cause by osmotic swelling.nnnMETHODSnCoiled sperm in semen from 439 infertile patients were quantified and their ultrastructure examined by electron microscopy. Hypo-osmotic swelling (HOS) and demembranation tests were performed to elucidate the nature of the coiling.nnnRESULTSnSemen from patients contained overall 3% of sperm with head-in-coil (HIC) and 8% other coiled forms, with 12% of patients having 20% or more such sperm. The percentage of coiled sperm (but not HIC) was correlated with age (R = 0.26, P = 0.003) and the epididymal secretory marker neutral alpha-glucosidase (R = 0.16, P < 0.001), and associated with heavy smoking and varicocele. Electron microscopy revealed coiling of tail filaments within the plasma membrane, resembling HOS. Some seminal coiled sperm and most sperm freshly coiled upon HOS could be opened by demembranation, while those that could not be opened were probably fixed in position by oxidation, which occurred more frequently in patients than semen donors.nnnCONCLUSIONSnSperm coiling in semen is common and independent of sperm quantity or hormonal status. Whereas HIC may have a genetic background, other coiled forms may be associated with a hostile endogenous milieu in the epididymis that causes swelling.

The Role of Potassium Chloride Cotransporters in Murine and Human Sperm Volume Regulation

Abstract Spermatozoa need to undergo regulatory volume decrease (RVD) upon ejaculation to counteract swelling due to the hypo-osmolality of female tract fluids. Defects in sperm RVD lead to failure in both cervical mucus penetration in humans and utero-tubal junction passage in mice. The role of K/Cl cotransporters (KCCs) in RVD was investigated by incubation of spermatozoa from the murine cauda epididymidis and from human ejaculates in media mimicking female tract fluid osmolalities in the presence of KCC inhibitors. Furosemide at 100 μM or more caused swelling of murine spermatozoa as detected with a flow cytometer by increased laser forward scatter over 30 to 75 min of incubation. Bumetanide, known to have low affinity for KCCs, was effective at 1 mM, whereas 10 μM and 20 μM of the specific inhibitor DIOA (dihydroindenyl-oxy alkanoic acid) increased cell volume. These drug doses were ineffective in human spermatozoa, which, however, responded to quinine, confirming the occurrence of RVD under control conditions. The molecular identity of the murine KCC isoform involved was determined at both mRNA and protein levels. Conventional RT-PCR indicated the presence of transcripts from Slc12a4 (KCC1), Slc12a6 (KCC3), and Slc12a7 (KCC4) in the testis, whereas RT-nested PCR revealed the latter two isoforms in sperm mRNA. Of these three isoforms, only SLC12A7 (KCC4) was detected in murine sperm protein by Western blotting. Therefore, besides organic osmolyte efflux and KCl release through separate K+ and Cl− ion channels, SLC12A7 also is involved in murine but not human sperm RVD mechanisms.

Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture

The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid α-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, β-hexosaminidase (N-acetylglucosaminidase) and α-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; β-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and β-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.

Transcytosis in the epididymis studied by local arterial perfusion.

SummaryThe transport of protein across the cells of the epididymal epithelium was studied using horseradish peroxidase. Transient vascular perfusion of the epididymis of the rat and golden hamster was achieved by pulsatile retrograde infusion into the testicular artery. Peroxidase was found in the interstitium and in the epithelium, located in vesicles, vacuoles and multivesicular bodies of principal, clear and apical cells. Similar findings were obtained in mice after systemic injection of the tracer. In the rat, discharge to the lumen was confirmed by the appearance of enzyme activity in luminal fluid from the caput epididymidis after local injection. The extent of transport amounted to no more than what has been considered leakage in physiological experiments, and the time-course of appearance complemented that found by electron microscopy. The level of transcytosis after pulsatile administration of peroxidase in vivo, as judged from the occurrence of tracer in the epithelium, was much less than that obtained during constant immersion in vitro. The protein was present in multivesicular bodies of principal cells and in vesicles of clear cells at short times after presentation in vitro, when it could not have arrived by endocytosis from the lumen. This suggests that routing of basal endocytic vesicles to the lysosomal apparatus occurs.

Regulation of the initial segment of the murine epididymis by dihydrotestosterone and testicular exocrine secretions studied by expression of specific proteins and gene expression

The murine caput epididymidis responded to deprivation of luminal fluid from the testis by regression of the initial segment but maintenance of the adjacent proximal and distal caput regions, as judged by immuno-histochemical staining of the glutamate transporter EAAC1 and the lipocalin MEP17 and enzymatic activity of β-galactosidase (β-Gal). Additional removal of circulating androgens by bilateral castration similarly led to loss of the initial segment and of the proximal caput but the distal caput was transformed into an epithelium containing more apical than principal cells staining for EAAC1; this epithelium resembled the precursor epithelium usually only seen in prepubertal juveniles. Administration of dihydrotestosterone (DHT) to the castrates maintained the proximal and distal caput epithelia and induced a proximal epithelium, which resembled the initial segment in its prominent staining for Golgi, EAAC1 and β-Gal activity, although it was short and exhibited no MEP17 expression. DHT was present in the c-ros knockout caput epididymidis lacking the initial segment and in the heterozygous organ but the DHT concentration was lower in the knockout corpus. The maintenance of the full complement of epithelia in the murine caput epididymidis in the adult thus requires a combination of luminal fluid from the testis, tissue DHT and the presence of the c-ros oncogene.