Absence Of Sperm Research Articles

Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia.

Infertility resulting from a severe defect in sperm production affects 2% of men worldwide. Of these men with azoospermia, the absence of sperm in semen, one in eight carry de novo deletions for a specific region of the Y chromosome. A candidate gene for the Y-chromosome azoospermia factor (AZF) has been identified and named Deleted in Azoospermia (DAZ). Here we describe the cloning and characterization of the Drosophila gene boule, which is a homologue of DAZ. The two genes encode closely related proteins that contain a predicted RNA-binding motif, and both loci are expressed exclusively in the testis. Loss of boule function results in azoospermia; meiotic divisions are blocked, although limited spermatid differentiation occurs. Histological examination of boule testes with cell-cycle markers indicates that the primary defect is at the meiotic G2/M transition. These results support the hypothesis that DAZ is the human AZF, and indicate that Boule and DAZ have an essential meiotic function in fly and human spermatogenesis.

Sperm Transfer in the Adélie Penguin

Sperm competition theory suggests that for a female that copulates with more than one male, the relative numbers of sperm inseminated will be important in determining which male fathers her offspring. Many studies of birds have reported insemination frequencies based on observations of apparent cloacal contact between male and female. Here we question whether cloacal contact is a reliable indicator of sperm transfer. This question has already been addressed in a small number of studies of captive species (Domestic fowl, Gallus domesticus: Penquite et al. 1930; Zebra Finch, Taeniopygia guttata: Birkhead et al. 1989; Bengalese Finch, Lonchura striata: Birkhead 1991). However Birkhead et al. (1988a) found differences in copulation behavior between wild and captive Zebra Finches, with wild birds copulating at a higher frequency than captive ones. So, although it has been shown for the above captive species that not all behaviorally successful copulations result in sperm transfer, it has yet to be shown for any wild species. Further, in these studies of captive species it was not determined whether sperm transfer failed as a result of the male's failure to ejaculate or absence of sperm in a successfully transferred ejaculate. Here we look at sperm transfer in a wild population of Ad6lie Penguins (Pygoscelis adeliae) by combining behavioral observations of cloacal contact and ejaculate transfer with collection of ejaculates and identification of presence or absence of sperm by microscopic examination. The aim of the study is to determine the true success rate, in terms of sperm transfer, of behaviorally successful copulations and the reason for failure of sperm transfer.

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17α-hydroxy-20β-dihydroprogesterone (17α20βOHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20β-hydroxysteroid dehydrogenase activity (20βHSD), testes homogenates were incubated with (3)H-labeled 17αOHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 17α20βOHP-(3)H, indicating that already immature testes contain 20βHSD activity and are able to produce 20β-reduced steroids. Second, 17α20βOHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 17α20βOHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20βHSD is present in testicular cells other than spermatozoa. Furthermore, 17α20βOHP is indeed secreted at a very early stage of testicular development; 17α20βOHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.

Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

Andrology: Percutaneous epididymal sperm aspiration for obstructive azoospermia

We report the use of percutaneous epididymal sperm aspiration as a simpler and more acceptable alternative to microscopic epididymal sperm aspiration for patients with obstructive azoospermia in whom bypass surgery is not feasible or has not been successful. Some contamination of the aspirate with blood is inevitable, but with careful sperm preparation techniques this can be reduced substantially in the final aliquot used for assisted conception. Spermatozoa with active forward progression may be used for gamete intra-Fallopian transfer treatment, but when this capacity is absent intracytoplasmic sperm injection is recommended. Three pregnancies were obtained in seven couples and a set of twins has been delivered.

Interpretation of Postmortem Vaginal Acid Phosphatase Determinations

Identification of evidence indicative of sexual assault, including elevated levels of acid phosphatase (AP) in the vagina, is an important part of the investigation of female homicidal deaths. In this study, vaginal AP levels in 43 female homicide victims, determined using sodium thymolphthalein as the substrate, were retrospectively correlated with semiquantitative assessment of spermatozoa in vaginal smears. The results were then compared with those from a prospectively studied age-matched group of controls. AP determinations ranged from 0 to 6,000 units (U)/L in the homicide group and from 1 to 726 U/L in the control group. In both groups, the majority of the AP values (homicide, 65%; and controls 85%) were < 100 U/L. Spermatozoa were identified on vaginal smears from 30% of the homicide victims and 17% of the controls. The spermatozoa-positive cases had AP levels that ranged from 2 to 6,000 U/L. In both groups, an AP value of > or = 400 U/L was always associated with the presence of spermatozoa on the vaginal smear. Intermediate AP values (100-399 U/L) were associated with the presence of spermatozoa in 60% of the control group and 22% of the homicide group; 18% of the homicide group and 3% of the control group were positive for spermatozoa despite AP values of < 100 U/L. In the absence of spermatozoa on a vaginal smear, an AP value of > 400 U/L strongly suggests the presence of semen that is either oligo- or aspermic. A low or intermediate AP determination, however, does not exclude the presence of semen and hence a careful search for spermatozoa on the vaginal smear is indicated, regardless of the AP value obtained.

Developmental expression of immobilin in the rat epididymis

Immobilin is a protein secreted by principal cells of the distal initial segment, intermediate zone and caput epididymidis of adult rats, which serves to immobilize spermatozoa. In the distal cauda, epithelial clear cells are involved in its endocytosis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of immobilin secretion and endocytosis in order to evaluate the testicular or epididymal factors which may influence or regulate immobilin expression. Our approach was to follow and compare the developmental expression of immobilin by light microscope immunocytochemistry in control and efferent duct ligated rats of different postnatal ages. Coincident with the morphological maturation of the principal cells by postnatal day 39, immobilin displayed the characteristic secretory immunostaining pattern found in adults. This adult-like expression occurred despite the absence of spermatozoa in the lumen but was coincident with high levels of circulating and luminal androgens. In contrast, immobilin secretion in rats whose efferent ducts were ligated at day 15 was weak to non-existent in the principal cells of the caput epididymidis at day 28 and remained so into adulthood, indicating that principal cells of this region of the epididymis are dependent either directly or indirectly upon testicular factors present in the lumen for immobilin expression. However, secretion of immobilin in the principal cells of the distal initial segment was unaffected by ligation and unlike the case in control rats high levels of immobilin also continued to be secreted into adulthood by the principal cells of the proximal initial segment. Thus in the distal initial segment immobilin secretion is not regulated by luminal factors originating from the testis, while in the proximal initial segment the normal suppression of immobilin that occurs by postnatal day 39 is. Despite ligation, endocytosis of immobilin by clear cells of the distal cauda epididymidis occurred by day 49, indicating that luminal testicular factors are not essential for stimulating the uptake of immobilin by these cells. The results taken together suggest that there are stimulatory and inhibitory luminal testicular factors involved in the regional development of immobilin secretion in the epididymis. There are also immobilin secreting regions in the epididymis, whose secretory development is independent of luminal testicular factors.

Seminal Vesicle Aspiration in the Management of Patients with Ejaculatory Duct Obstruction

Ejaculatory duct obstruction is a rare but significant cause of male factor infertility. Vasography is the current gold standard for the diagnosis of complete obstruction of the ejaculatory ducts. However, there is currently no reliable method to diagnose partial obstruction. We performed seminal vesicle aspiration under transrectal ultrasonographic guidance in 11 infertile men to assess the use of this diagnostic test in the evaluation and management of patients with ejaculatory duct obstruction. The absence of sperm within the seminal vesicle aspirate from 8 patients who had sperm in the ejaculate demonstrates that sperm are not normally present within the seminal vesicles. Numerous motile sperm were observed in the seminal vesicle aspirate from an azoospermic patient in whom vasography documented complete ejaculatory duct obstruction, demonstrating that sperm can reflux into the seminal vesicles in patients with distal obstruction. Two patients with suspected partial ejaculatory duct obstruction had sperm in the seminal vesicles. In conclusion, sperm are not normally present within the seminal vesicles and ejaculatory duct obstruction should be suspected in any patient with numerous sperm within the seminal vesicles.

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 microM external Ca2+ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 microM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur.

Results in the United States with Sperm Micro-Aspiration Retrieval Techniques and Assisted Reproductive Technologies

Improved methods of urological microsurgery and of various assisted reproductive technologies have resulted in the ability to achieve pregnancies with sperm aspirated microsurgically from men with bilateral congenital absence of the vasa deferentia or with ductal obstructions that cannot be surgically reconstructed. A survey was conducted of the results of such procedures performed in the United States during approximately 2 years. Female partner ovarian stimulation was initiated in 219 instances at 22 centers. Of the 219 procedures 23 (11%) were terminated without attempting any form of gamete fertilization or insemination of the wife for various reasons (no sperm or too few sperm retrieved, poor to absent sperm motility or poor quality eggs). The etiology of azoospermia in the 219 procedures was congenital absence of the vasa deferentia in 115 cases (52%), other congenital conditions in 15 (7%), failed vasectomy reversal in 37 (17%), infection in 4 (2%), other conditions (mainly ejaculatory dysfunctions that did not respond to electroejaculation and complex ductal obstructions) in 26 (12%) and not specified in 22 (10%). When only patient sperm was used pregnancy was achieved in 12 of 115 wives (10%) of those men who had congenital absence of the vasa deferentia and in 0 to 14% of the wives of men whose azoospermia was caused by other conditions. Of the 219 menstrual cycles in which ovarian stimulation was initiated pregnancy occurred in 24 (11%) with patient sperm and in 9 (4%) with donor sperm. Of these 33 pregnancies a full-term, live delivery occurred in 17 (52%), while 8 (24%) were ongoing at the time of reporting, 2 (6%) miscarried, 3 (9%) were chemical pregnancies, 1 (3%) resulted in a stillborn child and the outcome was unknown in 2 (6%). This new therapy offers only a modest chance of pregnancy. However, it affords men with azoospermia the opportunity to achieve paternity with their own sperm when other therapies are not available or applicable.

Characterization of a translocation and its impact on fertility in the pig.

A chromosome anomaly originally detected in two intersex pigs and previously reported as a Robertsonian translocation was characterized, and its transmission pattern was assessed by analyzing the progeny and immediate relatives of a sow. Cytogenetic investigations on four phenotypically normal relatives of the intersexes, including a sow, showed that the rearrangement was a reciprocal translocation involving the X chromosome and an autosome. Giemsa-banding and reverse-banding techniques indicated that the break in the X chromosome may have occurred in the terminal segment of the short arm (Xp) and that in the autosome in the proximal segment of the acrocentric chromosome 14 (14q). Reverse-banding techniques also revealed that the normal X is the late-replicating X in female translocation carriers. Centromere banding revealed an intercalary band on the long arm of the submetacentric chromosome representing the altered X in translocation carriers. The translocation was designated as rcp (X;14) (p+;q-). Chromosome analysis on 45 of 72 live offspring of the carrier sow showed a 20:25 distribution of carriers to normal piglets, with a carrier to normal ratio of 5:13 among females and 15:12 among male piglets, indicating an overall reduction of females (18) compared with males (27). Male carriers from one litter at sexual maturity showed hypoplastic testes and no spermatozoa in their ejaculates or fluid aspirates from the cauda epididymis. Their seminiferous tubules were narrow, spermatogenesis was impaired, and pyknotic and giant nuclei were abundant in the germinal epithelium. Meiotic preparations showed no stage beyond pachytene, suggesting that the absence of spermatozoa in the ejaculates may be due to the arrest of cells at the pachytene stage.

Induction of cyclooxygenase and prostaglandin E2 production by the bovine pre-embryo

A study was conducted to determine the induction of cyclooxygenase production in the cleaved and non-cleaved oocyte and to measure the secretion of prostaglandin E2 (PGE2), PGF2 alpha and tumour necrosis factor-alpha (TNF alpha) in the conditioned medium of non-mature oocytes (denuded), mature oocytes (oocyte-cumulus complex), cleaved oocytes, and non-cleaved oocytes (mature oocytes incubated in the absence of sperm). Cyclooxygenase was readily detectable in 48-h cleaved oocytes whereas the signal for the enzyme in the non-cleaved oocytes was weak or undetectable. Cyclooxygenase expression in the cleaved oocytes was transient and enzyme concentrations at the early morula stage (72 h) were low. Measurable amounts of both PGE2 and TNF alpha were secreted during the first 24 h of maturation and 6 h after fertilization, whereas PGF2 alpha was undetectable at these times. There was a dramatic enhancement in PGE2, PGF2 alpha and TNF alpha secretion 48 h after fertilization. At 72 h after fertilization, PGE2 concentrations were much lower and PGF2 alpha was again undetectable which corresponded to a low concentration of cyclooxygenase. In contrast, high TNF alpha activity continued to be detected even 72 h after fertilization. It is possible that the induction of the production of cyclooxygenase and the secretion of PGs and TNF alpha by fertilized oocytes at specific times after fertilization plays a role in the regulation of embryonic development, maternal immunological recognition of pregnancy, and the maintenance of a suitable hormonal environment for embryonic viability.

Double immunogold labelling demonstrating expression of recombinant genes for production of an anti-fertility vaccine

The multiple antibody technique for double immunogold labelling for the simultaneous localization of two antigens with negative staining was utilized to demonstrate the expression of recombinant genes in bacteria, with the primary antibodies being raised in different host species. For the production of a vaccine for immunological control of fertility, a multi-functional plasmid vector was introduced into the bacterium Pseudomonas aeruginosa containing the Dichelobacter nodosus fimbrial subunit gene with a grafted amino acid sequence of luteinizing hormone releasing hormone (LHRH) peptide. Fimbriae of this recombinant, when run on a SDC-polyacrylamide electrophoresis gel, gave a single broad band for LHRH peptide/ D. nodosus subunit and were harvested to produce the anti-fertility vaccine which, when injected into mice, produced atrophy of the testes with absence of sperm, resulting in reversible castration. Double immunogold labelling of the recombinant P. aeruginosa bacteria demonstrated fimbriae with strong expression of the LHRH-peptide, expression of the D. nodosus subunit and absence of host fimbriae.

Microsurgical Epididymovasostomy: Predictors of Success

The results of microsurgical epididymovasostomy for congenital and acquired vasoepididymal obstruction were retrospectively reviewed in 22 patients in an attempt to determine what preoperative or intraoperative factors might predict surgical success. The overall success rate, defined as sperm on postoperative semen analysis, was 48%. The presence of sperm on an intraoperative touch preparation from the epididymis was significantly correlated with response (chi-square 3.24, p <0.10) and no patient without sperm on touch preparation had sperm on subsequent semen analyses. Testicular biopsy positive for spermatogenesis and presence of motile sperm on intraoperative touch preparation were not statistically significant predictors of response. These results suggest that presence or absence of sperm on intraoperative touch preparation is the only significant prognosticator of successful microsurgical epididymovasostomy.

A balanced autosomal reciprocal translocation in an azoospermic bull

This appears to be the first reported case of a bull with a balanced autosomal reciprocal translocation associated with azoospermia. The analysis includes somatic chromosome banding, conventional meiotic analysis, and electron microscopy of synaptonemal complexes (SCs). The karyotype of the bull was found to be 60,XY,rcp(8;13)(q11;q24). Electron microscopy of SCs in microspread pachytene spermatocytes revealed a high incidence of terminal asynapsis of the smallest arm of the quadrivalent. Most quadrivalents with such asynapsis and few with nonhomologous synapsis showed associations with the XY sex bivalent, leading to complete meiotic arrest at late pachynema. Except for one diakinetic cell, no diplotene and subsequent stages were encountered in air-dried meiotic preparations. The presence of degenerating primary spermatocytes in SC preparations, as well as in testicular sections, and the absence of spermatozoa in ejaculates confirm the chromosomally derived male sterility of the bull. X-chromosome reactivation, evidenced by the cytomorphological reversal of associated sex bivalents, appeared to be the initial step in the degeneration of spermatocytes. Consequently, the formation of a separate, fully developed XY body, which was previously demonstrated on the periphery of spermatocyte nuclei in fertile bulls, could not be attained in this case. Extensive end-to-end association of autosomal bivalents in meiotically arrested, as well as degenerating, spermatocytes was a consistent and unique observation of this study. Such associations may lead to enhanced reactivation of the X chromosome.

Microsurgical vasovasostomy and epididymovasostomy for seminal tract obstruction: cause of obstruction and operative outcome

The surgical outcome in 42 patients with seminal tract obstruction who underwent microsurgical vasovasostomy or epididymovasostomy and were followed up for more than 3 months postoperatively is reported. Causes of the obstruction were vasectomy in 7 patients, childhood inguinal herniorrhaphy in 15, epididymitis in 5, Young's syndrome in 3 and others in 12. Twenty-eight patients had bilateral obstruction while 14 had unilateral obstruction. After 51 operations, including 11 reoperation in 9 patients, the anastomosis was patent in 83.3%, semen quality normalized in 21.4% and pregnancy was achieved in 31.0% of the 42 patients. Success rate of vasovasostomy and epididymovasostomy was 57.7% and 78.3%, respectively. Factors detrimentally affecting the operative outcome were obstruction due to Young's syndrome, obstruction for more than 10 years, absence of sperm in vasal fluid during the operation, elevated serum FSH levels and presence of seminal tract anomaly. Presence of serum antisperm antibodies had no effect on pregnancy rate. Microsurgical reanastomosis of the seminal tract resulted in restoration of fertility in many patients with seminal tract obstruction who had normal testicular spermatogenesis.

Detection of intravaginal spermatozoa after natural mating in the bitch.

Mismating is one of the most frequently presented reproductive problems presented to veterinarians. Confirming whether or not a mismating has occurred is necessary to determine if therapy will be instituted. Detection of spermatozoa in the vagina is irrefutable evidence of copulation; however, absence of sperm in a vaginal cytology cannot rule out coitus. In order to improve detection of spermatozoa post-coitus, a prospective study was initiated utilizing natural breedings of purebred beagles.

The fertility potential of male cystic fibrosis patients

Men suffering from cystic fibrosis (CF) are considered to be infertile because of azoospermia. In testicular biopsies from two patients with CF normal spermatogenesis was found despite the absence of sperm in the ejaculate. Fructose and prostaglandin were not detectable in the semen whereas the levels of acid phosphatase and zinc were within normal limits, indicating normal prostatic function and absence of seminal vesicles. These findings may improve the possibility for male patients with CF to father a child.

CLINICAL REVIEW

Failure of the normal ejaculatory mechanism may lead to retrograde ejaculation. This occurs most commonly as a result of transurethral prostatectomy, retroperitoneal lymph-node dissection and diabetic neuropathy. The diagnosis is based on the absence of sperm in the antegrade ejaculate and the presence of sperm in the post-masturbatory urine. If pharmacologic attempts to restore antegrade ejaculation fail, sperm recovery from the urine and intrauterine insemination are usually indicated. Surgical alternatives are discussed.

Microdose vasal injection of sodium fluoride in the rat

A single microdose (50 μg/50 μL) injection of sodium fluoride (NaF) into the vasa deferentia of adult male albino rats ( Rattus norvegicus) caused arrest of spermatogenesis and absence of spermatozoa in the lumina of the seminiferous tubules of the testes, which consequently led to a decline in the sperm count in the caudae epididymides. Scanning electron microscopy of cauda and vas deferens sperm revealed deflagellation and tail abnormalities. This is probably related to the alterations in the internal milieu of these organs which rendered the spermatozoa immotile and consequently caused fertility impairment in the experimental animals. Thus microdoses of sodium fluoride were found to affect reproductive function and fertility rate.