Evidence of aromatase localization in cytoplasmic droplet of human immature ejaculated spermatozoa

Lytochrome P450 aromatase is a microsomal enzyme catalyzing the conversion of androgens to estrogens. P450arom expression has been demonstrated in testicular and epididymal sperm cells of several species but very limited data have been reported about maturating human germ cells. In this study, human spermatozoa with cytoplasmic droplet anomaly have been utilized to investigate aromatase immunolocalization in the immature germ cells of human ejaculate. Immunodetection has utilized a polyclonal antiserum as primary antibody, a biotinylated IgG as secondary antibody and then the avidin-biotin-peroxidase complex amplification followed by the diaminobenzidine staining. A strong immunoreaction was observed in the cytoplasmic droplets retained around the midpiece of immature spermatozoa and also in the descending droplets of late maturing sperm, while the other cellular components were unstained. Therefore, this investigation has demonstrated, for the first time, aromatase immunolocalization in residual cytoplasm of human ejaculated sperm, suggesting cytoplasmic droplets as possible estrogen biosynthesis sites during human sperm differentiation.     

Apoptosis in human ejaculated spermatozoa

After the introduction of assays determining apoptosis in human ejaculated spermatozoa, several studies have been published about the relationship between apoptosis in spermatozoa and semen quality. Apoptosis in spermatozoa is significantly correlated with conventional semen quality parameters, but also with the outcome of assisted reproductive techniques. The apoptotic process is probably set in motion before ejaculation. Determining apoptosis in spermatozoa can improve selection criteria in assisted reproduction.     

Development of ER-alpha and ER-beta expression in the developing ovine brain and pituitary

Fetal neuroendocrine development in late gestation is critical for maintenance of fetal homeostasis, growth, and readiness for birth. We designed the present study to identify the regional patterns of expression of the two main isoforms of the estrogen receptor, ER-alpha and ER-beta, in the developing ovine fetal brain. Fetal (80, 100, 120, 130, and 145 days gestation), neonatal (1 and 7 days), and adult sheep were euthanized and the following tissues were collected: pituitary, hypothalamus, hippocampus, cerebral cortex, and brainstem. Both ER's are expressed in the ovine brain as early as 80 days gestation, and the expression of both receptors appears to be developmentally regulated. We conclude that both forms of the estrogen receptor are expressed in fetal brain and pituitary throughout the latter half of gestation.     

Detection of aromatase, androgen, and estrogen receptors in bank vole spermatozoa

Spermatozoa are highly specialized cells which transport a single-copy haploid genome to the site of fertilization. Before this, spermatozoa undergo a series of biochemical and functional modifications. In recent years, the crucial role of androgens and estrogens in proper germ cell differentiation during spermatogenesis has been demonstrated. However, their implication in the biology of mature male gametes is still to be defined. Our study provides evidence for the first time that aromatase, the androgen receptor (AR), as well as the estrogen receptors α and β (ERα and ERβ), are present in bank vole spermatozoa. We demonstrated the region-specific localization of these proteins in bank vole spermatozoa using confocal microscopy. Immunoreactive aromatase was observed in the proximal head region and in both the proximal and distal tail regions, whereas steroid hormone receptors were found only in the proximal region of the sperm head. Protein expression in sperm lysates was detected by Western blot analysis. Immunohistochemical results were analyzed quantitatively. Our results show that bank vole spermatozoa are both a source of estrogens and a target for steroid hormone action. Moreover, the presence of aromatase and steroid hormone receptors in the bank vole spermatozoa indicates a potential function of these proteins during capacitation and/or the acrosome reaction.     

Inhibitors of adenylate cyclase from ejaculated human spermatozoa

Adenylate cyclase from ejaculated human spermatozoa was inhibited by fluoride, Cu2+, Zn2+, Ni2+ and several carboxylic acids.     

Factors associated with estrogen receptors-alpha (ER-alpha) and -beta (ER-beta) and progesterone receptor abundance in obese and non obese pre- and post-menopausal women

There is scarce information about the factors associated with estrogen receptors (ER) at menopause. In 113 volunteers pre- and post-menopausal healthy women, grouped as with and without obesity, estrogen receptors-alpha and -beta, and progesterone receptor (PR) were measured by immunohistochemistry in skin punch biopsies obtained from the external gluteal area. In pre-menopausal women, biopsies and a blood sample were performed between days 7 and 14 of the cycle. Serum hormone levels were measured by immunoradiometric assay or radioimmunoassay. After menopause, ER and PR amounts decreased significantly. At pre-menopause, obese women had lower PR levels than non obese (P<.006). In the post-menopausal group, obese women showed higher ER-alpha (P<.03) and ER-beta (P<.02) levels than the non obese group. In the analysis of factors associated with the amount of steroid receptors for the total group, log[ER-alpha], log[ER-beta], and log[PR] were associated with age (P<.002, <.005, and <.004, respectively). The log[ER-alpha] was also associated with log[FSH] (P<.0008); meanwhile, the log[PR] showed a marginal correlation with log[FSH]. In pre-menopausal women no factor associated with any of the three receptors was found. In post-menopausal women log[ER-alpha] was associated with log[estrone] and log[DHEAS] (P<.003 and <.02, respectively). log[PR] was associated with BMI (P<.002), years since menopause (P<.05), and log[DHEAS] (P<.003). We concluded that ER and PR diminish sharply at post-menopause. At this stage the amount of receptors depends on several factors such as BMI, years since menopause, and androgen precursors.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

Ethanol extraction of basic proteins from ejaculated human spermatozoa

A method for the extraction of basic proteins from human ejaculated spermatozoa has been developed. It relies on the previously unreported observation that such basic protein is soluble in a solution containing 60% (v/v) ethanol. This unconventional method yields a high percentage of arginine-rich basic protein which is then able to be characterized on the basis of its amino acid composition. This method also allows comparisons to be made between single ejaculates by the banding pattern each displays when subjected to polyacrylamide gel electrophoresis.     

Morphological and biochemical analysis of cell death in human ejaculated spermatozoa.

We have analyzed the type of cell death occurring in human normal ejaculated spermatozoa. Sperm cells were prepared either by centrifugation alone (group 1) or by density gradient centrifugation (group 2) and were cultured for 24 hrs. Cells were examined after 4 and 24 hrs. By comparison unprepared spermatozoa were used as a control group. Necrosis was investigated by intra-cellular vital stain penetration and electron microscopy. Apoptosis was researched by DAPI staining, annexin V-binding, electron microscopy, DNA fragmentation and PARP cleavage. In group 1, after 4 hrs., there was a mixture of spermatozoa dead either by necrosis or apoptosis while after 24 hrs., necrosis was prominent. Similar findings were observed in the control group. In contrast, in group 2 apoptosis was the major form of cell death of spermatozoa after 24 hrs. of culture. These findings suggest that apoptosis can be an important factor when spermatozoa are used for assisted reproductive technologies.     

Immunolocalization of sperm protein 17 in human testis and ejaculated spermatozoa.

Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.     

Control of respiration and of motility in ejaculated bull spermatozoa

The relations between motility and respiration were studied in ejaculated bull spermatozoa respiring with lactate. Motility was quantitatively evaluated by a turbidimetric procedure as percentage of cells moving per minute from the bottom of the cuvette into the light path. For selective inhibition of ATP-consuming reactions including motility or of mitochondrial respiration, vanadate or cyanide, respectively, were used. Both inhibitors were found to produce proportional changes in motility and respiration. The simultaneous changes in motility and respiration were linked to shifts in the cellular ATP/ADP ratio. Partial uncoupling of respiration in vanadate-inhibited cells gave similar relations between respiration and ATP/ADP ratios as stepwise inhibition of ATP-utilizing reactions by vanadate. Presuming saturation kinetics with respect to the ATP/ADP ratio, half maximum constants of 1.7 and 4.7 for the ATP/ADP ratio and maximum values of about 130% and 300% (in comparison to untreated cells) were estimated for motility and respiration, respectively. Respiration showed a much steeper dependence on the ATP/ADP ratio than motility resulting in an apparent cooperativity coefficient of 2.9. From these dependences on the ATP/ADP ratio, the shares in the control of ATP turnover in untreated cells were estimated. At sufficient supply with substrate, more than 80% of control were excreted by motility and other ATP-utilizing reactions, the rest by mitochondrial ATP production, i.e., the reactions of oxidative phosphorylation.     

Activity of exoglycosidases in ejaculated spermatozoa of boar and bull

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of alpha-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? cytochemical studies

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen-DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P less than 0.01). Feulgen-DNA content significantly increased after capacitation in either B2 or follicular fluid (P less than 0.05, P less than 0.001 respectively), and so did sperm nuclear surface area (P less than 0.001). In this study we observed a negative correlation between Feulgen-DNA content and fertilization rate (P less than 0.02). Moreover, the greater effects on Feulgen-DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P less than 0.05) and Feulgen-DNA content higher (P less than 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.