Sperm-activating peptide type-V (SAP-V), a fifth member of the sperm-activating peptide family, purified from the egg-conditioned media of the heart urchin Brissus agassizii.

1. A novel type of sperm-activating peptide named sperm-activating peptide type-V (SAP-V) was isolated from the egg-conditioned media (egg jelly) of the heart urchin Brissus agassizii and the primary structure of the peptide was determined by fast atom bombardment mass spectrometry as follows: Gly-Cys-Glu-Gly-Leu-Phe-His-Gly-Met-Gly-Asn-Cys. 2. SAP-V and [Met(O)9]SAP-V stimulated the respiration of B. agassizii spermatozoa with half-maximal concentrations of 0.5 and 0.3 nM, respectively. However, half-maximal stimulation of the sperm respiration required 40 nM of S-carboxymethylated SAP-V. 3. SAP-V induced significant increases in the cyclic AMP and cyclic GMP levels in B. agassizii spermatozoa in a concentration-dependent manner. 4. The addition of SAP-V to B. agassizii spermatozoa resulted in a mobility shift of a major sperm protein (mol. wt from 133,000 to 129,000) on sodium dodecyl sulfate-polyacrylamide gels.     

Glutathione content in the semen of bulls of the lowland black-white breed

Glutathione concentration in the semen was determined in 113 ejaculates obtained from 30 bulls of the lowland black-white breed. The quantitative and morphological characteristics of the studied sperm samples indicated considerable individual differences but always within normal range. Glutathione content was determined in the plasma and in isolated spermatozoa by the method of Saville (Analyst, 1958, 83, 670). A high glutathione level in the spermatozoa (31 nmols in 10(9) cells) and in plasma (28.8 microM) was found. A positive correlation was demonstrated (p less than 0.05) between the level of the peptide in plasma and its level in spermatozoa. It was found that the glutathione level in spermatozoa was higher in the ejaculates with the cell count below the average level.     

Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep

This study was performed to determine whether oxytocin or vasopressin affect the transport of spermatozoa from the epididymis of rams in vivo. Under general anaesthesia, cannulae were inserted into each ductus deferens and passed into the cauda epididymis of 24 Oxford Down cross rams and the luminal fluid was collected at 10 min intervals for 2-3 h. Animals were divided into seven groups and received either (i) 2 ml 0.9% saline, (ii) 10 micrograms oxytocin, (iii) 100 micrograms oxytocin, (iv) 100 micrograms oxytocin antagonist, (v) 300 micrograms oxytocin antagonist followed by 100 micrograms oxytocin, (vi) 100 micrograms vasopressin, or (vii) 100 micrograms vasopressin followed by 100 micrograms oxytocin, all by i.v. injection. The mass of fluid and number of spermatozoa in each 10 min sample was measured and the motility of the spermatozoa was assessed. Treatment with saline did not affect the mass or the number of spermatozoa in the fluid collected. Oxytocin at 10 micrograms significantly increased both the output of fluid and the number of spermatozoa by twofold. Oxytocin at 100 micrograms produced a greater increase in both fluid output and the number of spermatozoa within 10 min of administration of the peptide. Treatment with oxytocin antagonist had no immediate effect, but subsequently caused a significant reduction in both fluid output and the number of spermatozoa. Pretreatment with oxytocin antagonist inhibited the stimulatory effect of oxytocin. Vasopressin did not increase the number or concentration of spermatozoa in the fluid and appeared to decrease fluid output. No significant changes in the morphology or motility of the spermatozoa collected was observed in any of the samples. These data demonstrate that oxytocin has specific actions on the epididymis to increase sperm transport. They indicate that local oxytocin may be involved in regulating basal contractility of the cauda epididymidis and that augmentation by the peptide in the peripheral circulation, as occurs around the time of ejaculation, may promote a significant increase in the transport of spermatozoa into the vas deferens and ejaculate.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

The effect of storage procedure at 5°C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72h. At 24h, sperm viability was higher for In-EPID (80.7±3.4%) than for the extended samples (44.8±2.9%, 37.7±3.9% and 48.6±6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.     

Secreted epididymal glycoprotein 2D6 that binds to the sperm's plasma membrane is a member of the beta-defensin superfamily of pore-forming glycopeptides

The plasma membrane of spermatozoa undergoes substantial remodeling during passage through the epididymal duct, principally because of changes in phospholipid composition, exchange of glycoproteins with epididymal fluid, and processing of existing membrane proteins. Here, we describe the interaction of an epididymal glycoprotein recognized by monoclonal antibody 2D6 with the plasma membrane of rat spermatozoa. Our goals have been to understand more about the mechanism of secretion of epididymal glycoproteins, how they interact with the sperm's plasma membrane, and their disposition within it. Reactivity to 2D6 monoclonal antibody was first detectable in principal cells in the distal caput epididymidis and as a soluble high-molecular-weight complex in the secreted fluid. It was not associated with membranous vesicles in the duct lumen. On cauda spermatozoa 2D6 monoclonal antibody recognized a 24-kDa glycoprotein (the subunit of a disulfide cross-linked homodimer of 48 kDa) that was present on the plasma membrane overlying the sperm tail. Binding of 2D6 to immature spermatozoa in vitro was cell-type specific but not species specific, and the antigen could only be extracted from cauda spermatozoa with detergents. Sequencing studies revealed that the 24-kDa glycoprotein was a member of the beta-defensin superfamily of small pore-forming glycopeptides of which several others (ESP13.2, Bin1b, E-2, EP2, HE2) are found in the epididymis. This evidence suggests that some epididymal glycoproteins are secreted into the luminal fluid in a soluble form and bind to specific regions of the sperm's surface via hydrophobic interactions. Given the antimicrobial function of beta-defensins, they have a putative role in protecting spermatozoa and the epididymis from bacterial infections.     

刺儿菜复合体的形态学研究

本文对不同产地,不同居群的全国２００份刺儿菜复合体标本的若干性状选用Ｅ．Ａｎｄｅｒｓｏｎ的形象化散点图和柱状图方法进行研究分析,认为将大刺儿菜与小刺儿菜分为独立的种是合理的。     

Atrial natriuretic peptide induces an acrosome reaction in giant panda spermatozoa and enhances their penetration of salt-stored porcine oocytes

Atrial natriuretic peptide (ANP) is a vasodilator peptide primarily produced in the heart. Locally synthesized ANP has been found in reproductive tissues of various mammals and humans, and plays an important role in rat oocyte maturation and human sperm function. The objective of the present study was to determine the effects of ANP on the function (acrosome reaction and zona penetration) of giant panda spermatozoa. In fresh and frozen-thawed spermatozoa that had been preincubated for 2.5h, treatment with ANP (for 60 min) significantly increased the proportion of acrosome-reacted spermatozoa; maximal response (an acrosome reaction in 18.3 and 21.8% of fresh and frozen-thawed spermatozoa, respectively) was detected at 1 nM ANP. Treatment with C-ANP-(4-23), an analogue of ANP and specific binder to natriuretic peptide receptors-C (NPRC), had no significant effect on the acrosome reaction. However, the cyclic guanosine 5'-monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor KT5823 completely abolished the effect of ANP on acrosome reaction. The effects of ANP, caffeine and heparin on frozen-thawed sperm function were studied by insemination of porcine salt-stored oocytes in a modified Tris-buffered medium (mTBM). The presence of ANP, caffeine or heparin in the insemination medium resulted in a higher proportion (P < 0.05) of oocytes with spermatozoa in the zona and perivitelline space (PVS), and a higher average number of spermatozoa/oocyte (P < 0.05) in the zona and PVS. However, in the absence of ANP, caffeine and heparin, there were no oocytes with a spermatozoon in the PVS. There were no differences among ANP, caffeine or heparin treatments for the proportion of oocytes penetrated or average number of spermatozoa/oocyte in the zona and PVS. In conclusion, we inferred that ANP induced the acrosome reaction of preincubated giant panda spermatozoa by a PKG pathway. Furthermore, ANP enhanced the penetrability of porcine salt-stored oocytes by frozen-thawed giant panda spermatozoa.     

The taxonomy and biology of Stenosiphonium Nees (Acanthaceae)

A taxonomic revision of Stenosiphonium Nees is provided. Morphological variation within the genus is documented, the relationship between Stenosiphonium and Strobilanthes Blume is discussed, and problems of species delimitation are resolved. Three species are recognized. Stenosiphonium cordifolium (Vahl) Alston is morphologically variable and is widespread throughout peninsular India and Sri Lanka. S. setosum T. Anderson and S. wightii Bremek. are restricted endemics from the southern Western Ghats, each known from very few herbarium collections. Both S. setosum and S.wightii are recognized as rare in accordance with IUCN criteria. There is evidence to suggest that all three species of Stenosiphonium are plietesial which may explain the paucity of herbarium collections of both S. setosum and S. wightii. The implications of a plietesial lie history strategy for the assessment of the conservation status of these species is considered.     

Evaluation of plasma membrane stability by detergent-induced rupture of osmotically swollen sperm.

A general assay for plasma membrane stability was developed and tested. Osmotically swollen spermatozoa were ruptured with detergents and their volume distribution was monitored with resistance pulse spectroscopy. The extent of cell breakage was determined and expressed as [D]50, the concentration of detergent necessary to lyse 50% of the initially intact cells. Preliminary experiments established the degree to which spermatozoa could be swollen without lysis (no detergent) and the ability of the method to detect known mixtures of intact and membrane disrupted spermatozoa. [D]50 values were determined for caput (immature) and cauda (mature) ram epididymal spermatozoa with four detergents (cetyltrimethylammonium bromide, sodium dodecylsulfate, Zwittergent 3-14, and sodium deoxycholate). [D]50 values for caput spermatozoa were higher than those for cauda spermatozoa (P less than 0.05) for all detergents but cetyltrimethylammonium bromide. These changes are consistent with a qualitative model of membrane structure and stability based on lipid shape and composition and with the compositional changes known to occur during epididymal maturation. Additional studies using rooster spermatozoa established that a typical cryopreservation protocol leaves the surviving spermatozoa with membranes with greater sensitivity to detergent-induced stress. Since osmotic swelling has been microscopically localized to the tail plasma membrane, the changes in membrane stability can be assigned specifically to that region.     

Effect of a fertilization-promoting peptide on the fertilizing ability and glycosidase activity in vitro of frozen-thawed spermatozoa in the pig

This study has evaluated the effect of fertilization-promoting peptide (FPP) on the fertilizing ability and glycosidase activity in vitro of frozen-thawed boar spermatozoa. Use of chlortetracycline (CTC) fluorescence analysis, as well as various glycosidase analyses and the oocyte penetration test showed that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. There were significantly (P < 0.05) more acrosome-reacted and penetrated in medium with 100 nM FPP than with 0, 50, 200 or 400 nM. The beta-N-acetylglucosaminidase (beta-GlcNAcase) activity was at least two-fold higher than other glycosidase regardless of FPP concentrations. In the same glycosidase, there were no differences in medium with different concentrations of FPP. The percentages of spermatozoa that reached acrosome reaction were affected by different periods (0, 1, 2, 3 or 4 h) of spermatozoa preincubation and were higher in medium with than without FPP. Penetration rates were decreased with preincubation periods of spermatozoa when oocytes were inseminated with spermatozoa preincubated in medium with and without FPP for the different periods. These rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 or 24 h. The activities of alpha-fucosidase, alpha-mannosidase, beta-galactosidase and beta-GlcNAcase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may have a positive role in promoting sperm function and glycosidase activity in the pig.     

The action of D2O on the motility of human spermatozoa.

The effect of 25%, 50%, 75% and 100% D2O on the survival of human spermatozoa was investigated. More spermatozoa were affected and died more quickly as the concentration of D2O increased. Spermatozoa died instantly in 100% D2O.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.     

蓟属两种植物的染色体研究

本文对蓟属（Ｃｉｒｓｉｕｍ）的两个形态相似的近缘种大刺儿菜和小刺儿菜进行了染色体研究,其中后者为首次报道。观察结果表明：两个种的染色体数目均为２ｎ＝２ｘ＝３４：它们的核型是：大刺儿菜．２ｎ＝２ｘ＝３４＝２０ｍ＋１２ｓｍ＋２ｓｔ：小刺儿菜．２ｎ＝２ｘ＝３４＝２２ｍ＋１０ｋｍ（２ＳＡＴ）＋２ｓｔ。通过核型比较,认为它们是两个独立的种．而且后者比前者进化。     

Demonstration of receptor-mediated chemotaxis by human spermatozoa. A novel quantitative bioassay

A novel in vitro technique is described for measuring the chemotactic activity of soluble substances for human spermatozoa. This new bioassay has demonstrated that the synthetic chemotactic peptide N-formyl-Met-Leu-Phe elicits a potent, specific (i.e., receptor-mediated) chemotactic effect on human spermatozoa with an EC50 of 3.2 X 10(-10) M. Quantitative chemotactic studies on human spermatozoa with nine N-formylated-peptide analogs have shown a rank order of peptide potency indistinguishable (p less than 0.001) from that obtained in binding and chemotactic studies with rabbit neutrophils. The competitive antagonist Boc (t-butoxycarbonyl)-Phe-Leu-Phe-Leu-Phe, 10(-6) M, completely inhibited the chemotaxis elicited by f-Met-Leu-Phe, 10(-9) M, and was able to shift by one order of magnitude the molar concentration required by f-Met-Leu-Phe-Phe and f-Met-Leu-Phe to elicit the maximal response. The ability of N-formylated peptides to function as sperm chemoattractants reveals a high degree of correlation with binding, chemotaxis, and lysosomal enzyme release previously employed to define the neutrophil chemotactic receptor. This first unequivocal demonstration of substances having a receptor-mediated chemotactic effect for human male gametes suggests that human spermatozoa may indeed have the ability to respond chemotactically to appropriate environmental signals.     

Fertilization in Sepia officinalis: the first mollusk sperm-attracting peptide

Egg mass extract was used to characterize regulatory peptides, involved in the successive steps of egg-laying of the cuttlefish Sepia officinalis. Among these peptides, a C-terminally amidated hexapeptide revealed a sperm-attracting activity. MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and Edman degradation led to a peptide of m/z 596.6 and the following primary sequence: Pro-Ile-Asp-Pro-Gly-Val-CO(NH2). From concentrations as low as 10(-17)M, the PIDPGVamide was able to attract freshly dissected spermatozoa. Nano-ESI-Q-TOF MS (nano-electrospray ionization-quadrupole-time-of-flight mass spectrometry) analysis established the quantitative occurrence of this peptide in different egg structures. The PIDPGVamide appears to be synthesized in oocytes during vitellogenesis and released by the embedded oocytes in the external media during egg-laying to facilitate fertilization by increasing chances of gamete collision. This novel peptide called SepSAP for Sepia sperm attracting peptide is the first sperm-attracting peptide, identified in mollusks or even in protostomians.     

Novel identification of peripheral dopaminergic D2 receptor in male germ cells

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology.