Ovarian follicular superstimulation and oocyte maturation in the anoestrous southern hairy-nosed wombat, Lasiorhinus latifrons.

This study investigates the effect of three exogenous gonadotrophin regimens on ovarian follicular development in southern hairy-nosed wombats during the non-breeding season. Females were given either porcine follicle stimulating hormone (pFSH; total of 200 mg at 12 h intervals over 7 (Group 1), or 4 days (Group 2)), or pregnant mares' serum gonadotrophin (PMSG; single dose of 150 I.U. (Group 3)). In all treatment groups 25 mg of porcine luteinising hormone (pLH) was used to trigger maturation; Groups 1 and 2 received pLH 12 h after the final pFSH injection and Group 3 received pLH 72 h after PMSG. The results showed Group 1 produced significantly more follicles per ovary (5.91+/-1.28) than Group 2 (1.67+/-0.62), or Group 3 (2.17+/-1.16) at p<0.05. Control females received saline injections concurrently with the three treatment groups (n=6; 2 control animals for each treatment group). No follicular development occurred in any control female. Analysis of oocyte nuclear status revealed that while oocytes from all three treatment groups had resumed meiosis, only those in Group 1 (7-day pFSH/pLH treatment) progressed to metaphase II. These results have implications for the development of assisted breeding strategies in this species.

Pouch young removal and return to oestrus in wild southern hairy-nosed wombats (Lasiorhinus latifrons).

The southern hairy-nosed wombat (Lasiorhinus latifrons) is a seasonal breeding, burrowing marsupial adapted to a semi-arid environment and the closest relative of the endangered northern hairy-nosed wombat (Lasiorhinus krefftii). Females typically give birth to one to two young every 3 years with young weaned at 360-400 days. This study examined the occurrence of polyoestry in a wild population of southern hairy-nosed wombats, and in particular the ability of this species to produce additional offspring in the same breeding season if a young was prematurely lost or removed. Pouch young were removed during the breeding seasons of 1996/1997 and 2003. No females from the 1996 (n=3)/1997 (n=3) group gave birth to a second pouch young in the same breeding season. However, two females in this group gave birth to young the following season. In contrast, all the 2003 group of females (n=6) produced a second offspring in the same breeding season after removal of pouch young (RPY). The reason for the different response to RPY between the two groups is unknown. These studies confirm that southern hairy-nosed wombats are polyoestrus in the wild and are capable of producing more than one offspring in a single breeding season. Females that failed to return to oestrus in the breeding season that pouch young were removed bred again in the following season. Rapid replacement of southern hairy-nosed wombat pouch young in the same breeding season as RPY suggests that this procedure, linked to either hand-rearing or interspecific cross-fostering, should be seriously considered as a priority conservation action to increase the population size of the critically endangered sister species, the northern hairy-nosed wombat.

Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials (Phascogale tapoatafa) that survive beyond the breeding season in captivity.

The semelparous dasyurids display a unique life history, in that all males die within a few weeks of the completion of the breeding season. Studies of several semelparous species have revealed that the male die-off is stress-related, and accompanied by increased plasma androgen and cortisol levels and decreased corticosteroid binding capacity, resulting in suppression of immune and inflammatory responses. This study examines the endocrine profile of male brush-tailed phascogales (Phascogale tapoatafa) that survive beyond the breeding season in captivity. Plasma cortisol, corticosteroid binding globulin and albumin levels were monitored in both males and females and steroid partitioning calculated. Captive males surviving beyond the breeding season did not show the elevation in plasma cortisol and decrease in corticosteroid binding capacity reported in wild males. Plasma albumin concentrations also remained constant during the sampling period. These data indicate that captive males do not undergo the same stress response described in wild populations.

Changes in semen quality and morphology of the reproductive tract of the male tammar wallaby parallel seasonal breeding activity in the female.

Changes in semen quality and morphology of the male reproductive tract were studied throughout the year in the highly promiscuous tammar wallaby. Body size, semen quality and gross morphology of the reproductive organs were assessed in adult males each month from January to November. The mean weight of males was similar in most periods sampled, but males were slightly heavier in the minor (P < 0.05) than the non-breeding season. Since body weight was correlated with weights of the testes, epididymides and accessory sex glands, organ weights were adjusted for body weight in subsequent analyses. In the major breeding season (late January/early February), when most females go through a brief, highly synchronized oestrus, the testes, prostate, Cowper's glands, crus penis and urethral bulb were heaviest, volume and coagulation of ejaculates were greatest, and sperm motility had increased. Semen samples collected by electroejaculation at this time contained low numbers of spermatozoa, possibly as a result of dilution and entrapment by the seminal coagulum or depletion of epididymal stores during intense multiple mating activity. In the non-breeding season (late May-July), when mating does not normally occur in the wild, there was a significant decrease in the relative weight of nearly all male reproductive organs and a decline in most semen parameters. In the minor breeding season (September-November), when pubertal females undergo their first oestrus and mating, the weights of testes, epididymides and most accessory sex glands had significantly increased similar to those of males in the major breeding season. The total number and motility of ejaculated spermatozoa were highest during this period, but the volume and coagulation of ejaculates and weight of the prostate had only increased to levels that were intermediate between the major and non-breeding seasons. Ejaculate volume was strongly correlated with prostate weight, and % motile spermatozoa was strongly correlated with epididymis weight. Semen quality thus varied seasonally with changes in androgen-dependent reproductive organs in the male tammar wallaby and appeared to be influenced by the seasonal timing of oestrus in females. Semen quality may also improve in response to an increase in the number of available oestrous females.

Paternity success and the direction of sexual selection in a field population of a semelparous marsupial, Antechinus agilis.

Antechinus agilis is a small sexually size dimorphic marsupial with a brief annual mating period of 2-3 weeks. All males die after this period, and females give birth to up to 10 young. Mating is thought to be promiscuous, however, there is no field data to confirm this. Using microsatellites, we investigated paternity patterns over two seasons in a wild population. Male weight was significantly positively related to the number of females fertilized and with the number of offspring sired, in both years. Furthermore, selection gradients indicated selection for larger males. Both results suggest that size dimorphism in A. agilis can be explained by sexual selection for larger males. The proportion of offspring sired within litters, did not relate to male size. Therefore, larger males are more successful through higher mating access, not through their sperm outcompeting that of smaller males. As expected from their known ranging behaviour, the number of offspring within litters left unassigned to a father did not depend on the grid location of the mother. Female size did not differ between successful reproducing and unsuccessful females. However, females that weaned offspring had larger heads than females that did not wean offspring. Males did not 'prefer' mating with larger females, nor did assortative mating occur. From our results, the mating system of A. agilis is clearly promiscuous. Selection for larger males occurred in both years, even though in one year the operational sex ratio was highly female biased, suggesting that the potential reproductive rate is a better predictor of the direction of sexual selection in A. agilis.

Determination of seasonality in southern hairy-nosed wombats (Lasiorhinus latifrons) by analysis of fecal androgens.

Little is known about the reproductive biology of Australia's critically endangered northern hairy-nosed wombat (Lasiorhinus krefftii), largely due to its cryptic nature and the difficulty in accessing the small remaining population of about 70 animals. Using the noninvasive technique of fecal steroid analysis, we have examined the endocrinology of the more common yet closely related southern hairy-nosed wombat (Lasiorhinus latifrons). The aims of this study were to 1) develop and validate fecal androgen analysis in this species, 2) examine and compare seasonal differences in fecal and plasma androgens in male wombats, and 3) correlate seasonal differences in androgens with changes in male accessory glands (prostate and bulbourethral gland). Fecal androgens were extracted in ether; concentrated; separated by HPLC into testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) fractions; and quantitated by RIA. The concentrations of androgens in fecal pellets from 14 wild southern hairy-nosed wombats as determined by RIA varied over the range 6.6-25.0 ng/g dry weight for T, 4.0-24.2 ng/g dry weight for DHT, and 0-34.8 ng/g dry weight for Adiol. For each androgen, a highly significant linear correlation was observed between plasma and fecal concentrations. When individuals were grouped into either breeding season (pellets collected between August-November) or nonbreeding season (collected between February-April), significant (P < 0.05) differences between seasons were observed for both plasma and fecal T, plasma DHT, and fecal Adiol. For all androgens, the mean fecal and plasma concentrations were higher during the breeding season than the nonbreeding season. A significant (P < 0.001) correlation was observed between fecal T and prostate weight, while DHT and Adiol correlations were nonsignificant. Significant correlations were observed, however, between all three fecal androgens and bulbourethral gland weight. These studies demonstrate that fecal T is a valid indicator of reproductive status in the male southern hairy-nosed wombat, with significant correlations observed between fecal T, plasma T, and prostate and bulbourethral gland weights. These findings have important implications for the study of the reproductive endocrinology of the critically endangered northern hairy-nosed wombat.

Sperm transport and storage in the agile antechinus (Antechinus agilis).

This study was an examination of the timing of ejaculation and the dynamics of sperm transport in the female reproductive tract of the agile Antechinus (Antechinus agilis) and the relationship of these parameters to single and multiple matings. Mating in this species is characteristically long compared with that of other mammals, lasting for up to 8-12 h during which time the pair remain locked together. After the first hour of mating, only approximately 40% of males had ejaculated, but by the third hour all males had ejaculated. The total number of spermatozoa extracted from the female tract remained at approximately 30 x 10(3) spermatozoa per side over the next 9 h of copulation. After completion of male/female access (12 h), approximately 56% of spermatozoa extracted were located in the lower isthmic region of the oviduct where specialized sperm storage crypts are located. The number of spermatozoa extracted from the female reproductive tract did not decline over the next 3 days, but there was a change in the distribution of spermatozoa with an increase in the proportion of extracted spermatozoa stored in the lower isthmus (approximately 76%). However, 7 to 14 days after mating, only approximately 30% of the stored spermatozoa ( approximately 9.4 x 10(3) spermatozoa per side) were still present in the isthmus. When females were mated with a second male on a consecutive day, the sperm numbers extracted from the tract were about twice that deposited during single mating, with sperm transport to the lower isthmus occurring over a similar time frame. Although the occurrence of extended copulations in the wild has not yet been confirmed, these laboratory results suggest that similar periods of copulation are likely, since completion of the ejaculation process requires at least 3 h. The extended copulation in A. agilis reduces the possibility of an early second mating, which might interfere with the normal transport and crypt colonization of spermatozoa through competition.

Timing of mating, sperm dynamics, and ovulation in a wild population of agile Antechinus (Marsupialia: dasyuridae).

Timing of mating, sperm transport and storage, and ovulation were examined in a wild population of agile Antechinus (Marsupialia: Dasyuridae) in order to ascertain the validity of direct comparisons between captive and field-based mating studies in this species. Mating commenced in early August, and all females had ovulated by the 27th of the month. Fifty-five percent of the mated females caught that had not yet ovulated were captured on 19-20 August. This corresponded with a peak (67%) in the ovulation date determined for pregnant females. Approximately 25.9 x 10(3) spermatozoa per side were recovered from the reproductive tract of each mated female captured (range: 1.7 x 10(3)-75.5 x 10(3) spermatozoa per side). Spermatozoa were consistently found in greater numbers in the lower isthmus (19.7 x 10(3) +/- 19.9 x 10(3) spermatozoa per side) of the oviduct ( approximately 67% of all sperm found in the female tract; range 17-94%) than elsewhere in the reproductive tract. Few spermatozoa were found in the upper isthmus, and none were detected in the ampulla. Sperm number in the female reproductive tract supports the hypothesis that females will mate several times within the one estrus. At the conclusion of the rut, approximately 80.0 x 10(3) spermatozoa remained in each testis and approximately 630 x 10(3) spermatozoa in each epididymis. Most epididymal spermatozoa were restricted to the distal corpus/proximal cauda regions of the duct. This study shows that both field and laboratory reproductive data correlate well in the agile Antechinus and that successful breeding is indeed an exercise in reproductive brinkmanship.

Isolation and characterization of rat sperm tail outer dense fibres and comparison with rabbit and human spermatozoa using a polyclonal antiserum.

Rat outer dense fibres were isolated from cauda epididymal spermatozoa using mechanical and chemical dissection methods. Sperm tail isolation procedures were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated outer dense fibres revealed at least nine Coomassie brilliant blue stained bands, and 12 silver staining bands. The most abundant proteins were a large band between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolated proteins showed similar compositions, being abundant in aspartic and glutamic acid, serine, glycine and leucine. However, the content of cysteine and proline was highly variable among the isolated proteins. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat outer dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum also showed crossreactivity in the perforatorium of rat sperm heads and the acrosome and equatorial segment of rabbit sperm heads. These data indicate that it is technically possible to isolate proteins from the outer dense fibres that will enable further studies of the amino acid sequences of sperm tail proteins.

Field biology of the platypus (Ornithorhynchus anatinus): historical and current perspectives.

The field biology of the platypus, Ornithorhynchus anatinus, was first studied by a number of expatriate biologists who visited the Australian colonies to collect specimens in the 1800s. Their work was followed in the early to mid-1900s by a group of resident natural historians and later by an increasing number of academic biologists. All of these workers contributed significantly to the current understanding of the field biology of this unique Australian species. The platypus occupies much the same general distribution as it did prior to European occupation of Australia, except for its loss from the state of South Australia. However, local changes and fragmentation of distribution due to human modification of its habitat are documented. The species currently inhabits eastern Australia from around Cooktown in the north to Tasmania in the south. Although not found in the west-flowing rivers of northern Queensland, it inhabits the upper reaches of rivers flowing to the west and north of the dividing ranges in the south of the state and in New South Wales and Victoria. Its current and historical abundance, however, is less well known and it has probably declined in numbers, although still being considered as common over most of its current range. The species was extensively hunted for its fur until around this turn of this century. The platypus is mostly nocturnal in its foraging activities, being predominantly an opportunistic carnivore of benthic invertebrates. The species is homeothermic, maintaining its low body temperature (32 degrees C), even while foraging for hours in water below 5 degrees C. Its major habitat requirements include both riverine and riparian features which maintain a supply of benthic prey species and consolidated banks into which resting and nesting burrows can be excavated. The species exhibits a single breeding season, with mating occurring in late winter or spring and young first emerging into the water after 3-4 months of nurture by the lactating females in the nesting burrows. Natural history observations, mark and recapture studies and preliminary investigations of population genetics indicate the possibility of resident and transient members of populations and suggest a polygynous mating system. Recent field studies have largely confirmed and extended the work of the early biologists and natural historians.

Development and use of surgical procedures to bypass selected regions of the mammalian epididymis: effects on sperm maturation.

Microsurgical procedures have been used to examine the function of selected regions of the epididymis. Vasoepididymostomy in the rabbit and rat confirmed that spermatozoa require some exposure to the epididymal environment for normal development of motility and fertilizing ability. In both of these species surgical redirection of spermatozoa from a region just distal to the initial segment into the vas deferens resulted in a significant reduction in sperm viability, motility and fertility compared with untreated animals. Surgical bypass of the distal cauda epididymidis in the rabbit did not significantly reduce the number of spermatozoa in ejaculates or the total sperm output accumulated during exhaustive ejaculations. The capacity of these bypass animals to store spermatozoa may be explained by the post-surgical formation of convolutions in the normally straight segment of the rabbit vas deferens. Vas autografts in the rabbit and rat were used to bypass selected epididymal segments. In rabbits, vascularized grafts were used as bridges to bypass the distal caput and proximal corpus epididymidis. Despite high rates of graft survival and patency in this species, the motility and viability of ejaculated spermatozoa were similar to those observed following high level vasoepididymostomy. In rats, non-vascularized vas grafts also showed a high survival and patency rate. There was a transitory reduction in sperm viability and motility 2 weeks after surgery which was associated with structural changes, especially in the epithelium, in vas bridge autografts during revascularization. Longer term observations (3 and 6 months) showed recovery of epithelial morphology and by three months after surgery, no difference was observed in sperm motility, sperm viability or fertility between experimental groups and controls. Similar results were obtained when epididymoepididymostomy was used as the surgical procedure to bypass the same regions of the epididymis. The evidence considered in this review clearly shows that the proximity of vasoepididymal bypass to the testis is inversely related to the state of maturity of ejaculated spermatozoa. More surprisingly, perhaps, it reveals that exposure of spermatozoa to the distal caput and proximal corpus regions of the epididymis in the rat is not obligatory for spermatozoa to develop a level of maturation in the remaining regions of the epididymis sufficient to ascend the female tract and fertilize ova. This finding suggests that the epididymis is more flexible in its functions than had been considered previously and that sperm maturation may not be as dependent on specific regional functions of the epididymis as had been previously proposed.

The role of the epididymis in human infertility.

This review describes the role of the epididymis in human infertility, by analysing the results of epididymovasostomies which confirm that the more distal the site of obstruction the greater the chance of fertility. The use of epididymal spermatozoa for in vitro fertilization (IVF) yielded poor results in contrast to intracytoplasmic sperm injection using either epididymal or testicular spermatozoa. The nature of the pathology causing obstructive azoospermia is examined reviewing in particular the possible role of mercury toxicity in Young's syndrome. This review describes the results of studies that show that the level of obstruction within the epididymis is correlated with the presence of sperm antibodies and distal obstructions are associated with the presence of sperm antibodies. The demonstration that necrozoospermia decreases with increased sperm transport through the epididymis, when combined with the observation of normal testicular sperm morphology, represents the basis for the disorder termed epididymal necrozoospermia. However, to date the nature of the epididymal pathology causing this disorder remains obscure.

Isolation and characterization of human and rabbit sperm tail fibrous sheath.

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.

Frozen-thawed epididymal spermatozoa for intracytoplasmic sperm injection.

OBJECTIVE: To report the fertilization rate and pregnancy results for intracytoplasmic sperm injection (ICSI) cycles using frozen-thawed epididymal spermatozoa. DESIGN: Retrospective analysis of consecutive ICSI cycles. SETTING: Tertiary referral center for infertility. PATIENT(S): Infertile couples in whom 39 patients (59 ICSI cycles) required the use of frozen-thawed epididymal spermatozoa. As no cycles were performed using fresh epididymal spermatozoa, outcomes were compared with those of 130 couples (170 ICSI cycles) using fresh ejaculated spermatozoa for severe oligoasthenozoospermia-teratozoospermia, in which < 200,000 motile spermatozoa were retrieved after Percoll density gradient centrifugation. INTERVENTION(S): Epididymal sperm aspiration during microsurgery, followed by ICSI. MAIN OUTCOME MEASURE(S): Fertilization, embryo cleavage, ET, and clinical pregnancy rates (PRs). RESULT(S): A total of 484 metaphase II oocytes were injected with frozen-thawed epididymal spermatozoa, resulting in a two-pronuclear fertilization rate per injected metaphase II oocyte of 47%, significantly lower than in cycles using fresh ejaculated spermatozoa (73%). Embryo implantation rates (12.0% versus 13.3%) and clinical PRs per transfer (18.4% versus 27.0%) were not different. When the prefreeze vitality was > 20%, compared with lower vitality, both the fertilization (56% versus 22%) and embryo cleavage (91% versus 57%) rates were significantly greater. CONCLUSION(S): The routine collection and cryopreservation of epididymal spermatozoa at microsurgery allows multiple ICSI treatment cycles with success rates similar to those of ejaculated spermatozoa. However, when the vitality of aspirated spermatozoa is < 20%, the poor fertilization rates indicate the need to consider an alternative source of viable spermatozoa.

Effect of cooling and cryopreservation on sperm motility and morphology of several species of marsupial.

The effects of long-term cooling and freezing on sperm motility are described for six marsupial species: the fat-tailed dunnart, koala, brushtail possum, long-footed potoroo, northern brown bandicoot and ring-tailed possum. The effects of up to eight days of cooling at 4 degrees C on the motility of dunnart spermatozoa and the effect of cryopreservation on spermatozoa of the other species were determined. The cryoprotectant used was a Tris-citrate-fructose-egg yolk-glycerol diluent. The percentage and rating of sperm motility, and sperm structure, as determined by light microscopy, were investigated. Sperm motility in the fat-tailed dunnart was retained for up to six days when cooled to 4 degrees C, suggesting that sperm from this species have some degree of tolerance to cold shock. After this time, however, the percentage of motile spermatozoa and their motility rating declined. In all species except the fat-tailed dunnart, reinitiation of motility following cryopreservation occurred across a range of glycerol concentrations (4-17%). Cryoprotectant containing 6% and/or 8% glycerol resulted in little change of motility rating or of the percentage of live sperm after thawing, although there was some decline in the percentage of motile sperm. The unusual structural and motility characteristics of dunnart spermatozoa may account for the lack of success of sperm cryopreservation in this species.

DNA fingerprinting to determine paternity in laboratory rat sperm competition experiments.

Prior to this study a significant amount of research had been undertaken in the field of sperm competition in mammals. However, males of different strains have been required in each of these studies to enable paternity assignment through gene expression, which has consequently resulted in problems with differential fertilising capacity being encountered. In this study paternity assignment of progeny from sperm competition experiments with Sprague Dawley rats was achieved by multilocus DNA fingerprinting using band locus matching of individual specific banding patterns between progeny and parents. Trials with 4 restriction enzymes and 5 digoxygenin labelled probes (4 oligonucleotide and 1 cloned) achieved the highest levels of DNA fingerprint heterozygosity using AluI(CAC)5 and HinfI(CAC)5 combinations; however, paternity could not be determined in all offspring, due to a higher than expected degree of inbreeding within the rat population used in this study. This was demonstrated in subsequent comparisons of genetic diversity of three laboratory rat breeding populations from two different animal breeding facilities. Data from the rat mating study showed that, under conditions of direct sperm competition, second males given access to a mated oestrus female either 0.5 or 6.0 h after the first mating consistently required less time than the first to ejaculate: 7.6 min vs. 19.5 min (0.5 h delay ); 7.8 min vs. 19.5 min (6.0 h delay). A second males siring advantage was identified using DNA fingerprinting in both delay groups for those offspring on which paternity could be determined: 0.5 h delay, 1st = 39%, 2nd = 61%; 6 h delay, 1st = 34%, 2nd = 66%.

Pregnancies after microsurgical correction of partial epididymal and vasal obstruction.

A group of 16 infertile patients suspected of having a partial epididymal obstruction on the basis of severe oligozoospermia, normal-sized testes and a normal serum follicle-stimulating hormone, underwent scrotal exploration. Evidence of partial obstruction of the epididymis was found in 13 cases and of the vas deferens in one case, and was supported by finding normal spermatogenesis on testicular biopsy. Vasoepididymostomy or vasovasostomy were performed, resulting in a significant improvement of semen analysis in 50% of cases and in six pregnancies in two patients. The diagnosis of partial epididymal obstruction should be considered when the above criteria are met. If pregnancies do not result when intracytoplasmic sperm injection (ICSI) is used with the ejaculated spermatozoa, a testicular biopsy followed by a microsurgical by-pass procedure should be considered whenever normal spermatogenesis is diagnosed. In all cases, the epididymal spermatozoa should be aspirated during the operation and either used immediately for insemination or stored frozen. The remarkable results of the new artificial reproduction technologies and in particular ICSI, question the indication for microsurgical correction in cases of partial epididymal obstruction.

Isolation and partial characterization of rat sperm tail fibrous sheath proteins and comparison with rabbit and human spermatozoa using a polyclonal antiserum.

Rat sperm tail fibrous sheath was isolated using mechanical and chemical dissection methods from spermatozoa collected from the cauda epididymis. The procedures used to isolate the fibrous sheath were monitored by phase-contrast microscopy and purity was verified by electron microscopy. SDS-PAGE of isolated total fibrous sheath revealed at least 17 bands when stained with Coomassie brilliant blue and 20 bands with silver stain. The most intensely staining proteins, using both staining methods, were a double band at 80-87 kDa, and a band at 28.5 kDa, whereas with silver staining, bands at 66.2 kDa and kDa were also intensely stained. Electroelution following SDS-PAGE was used to isolate 11 of these proteins (116.4, 87.5, 80.9, 66.2, 57.2, 49.7, 46.8, 37.3, 32.7, 28.5 and 15.5 kDa). Amino acid analysis revealed that these proteins were abundant in aspartic and glutamic acid, glycine, serine and leucine, while histidine and phenylalanine were of low abundance. The content of cystine varied widely from 9.4% to 1.4%. The amino termini of the 80.9 kDa, 32.7 kDa, 28.5 kDa and 15.5 kDa proteins were blocked. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat fibrous sheath was localized to the principal piece of the rat, rabbit and human spermatozoa, but in the rabbit it also labelled the equatorial region of the head. Western blotting detected all protein bands in isolated fibrous sheath and a similar range of proteins in the spermatozoa of rat and rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)

Fertility in cases of hypergonadotropic azoospermia.

OBJECTIVE: To review the outcome of reconstructive microsurgery in men with hypergonadotropic azoospermia. DESIGN: A retrospective study on patients with hypergonadotropic azoospermia who underwent scrotal exploration and bypass microsurgery. SETTING: Male infertility microsurgery clinic affiliated with a tertiary university hospital. PATIENTS: Thirty-one hypergonadotropic azoospermic men with evidence of spermatogenesis on testicular biopsy. INTERVENTIONS: Microsurgical exploration of the testes, operative sperm aspiration, and bypass procedures: vasoepididymostomy or vasovasostomy. MAIN OUTCOME MEASURES: Intraoperative aspirated sperm, postoperative ejaculated sperm, proof of fertilizing ability (fertilizations), and pregnancies. RESULTS: Sperm were aspirated intraoperatively in all cases and were detected in postoperative ejaculations in 87%. Of the 14 patients with long-term follow-up data, 6 achieved pregnancies (8 children), and 3 more demonstrated the capacity for fertilization at IVF. CONCLUSIONS: The observation of a high serum FSH in men with azoospermia does not rule out the possibility of obstruction and the capacity for fertility. Caution should be exercised particularly if unilateral testicular atrophy is present. A testicular biopsy should be performed to detect possible spermatogenesis and, if present, then a microsurgical bypass can lead to a successful pregnancy.

Comparative structure and function of marsupial spermatozoa.

Marsupial sperm structure has been the focus of many comparative studies in the last 30 years. Although the basic organization of the marsupial spermatozoon is similar to that of eutherian mammals, spermatozoa from this branch of therian evolution have developed a specific suite of characters which clearly distinguish them from the Eutheria. This review surveys these specializations and examines current knowledge on their respective functions and the forces which shaped their evolution. Nuclear shaping and stability, the asymmetric positioning of the acrosome, and the unusual neck articulation are discussed. Although recent observations have provided evidence of a marsupial equatorial segment and posterior ring, the marsupial equivalent of the eutherian postacrosomal sheath has not been identified. The unusual neck structure of marsupial spermatozoa and the mobile articulation of the connecting piece are discussed in relation to nuclear rotation and the events associated with this process. Increasing flagellar length in some species is associated with extremes in flagellar organization and its effect on sperm motility is discussed.