Large Numbers Of Sperm Research Articles

Enhancement of spermiation in European sea bass (Dicentrarchus labrax) at the end of the reproductive season using GnRHa implants

Abstract Spermiating European sea bass ( Dicentrarchus labrax ) were treated at the end of the reproductive period (22 February 2002) with implants of gonadotropin-releasing hormone agonist (GnRHa), in order to examine their potential in enhancing expressible milt volume, sperm quality and in vitro fertilization success. The GnRHa implants at all doses (20, 40 and 80 μg GnRHa kg −1 body weight [bw]) stimulated a significantly higher production of milt compared to controls over a 27-day period, increasing total volume from 0.5 to 2.2 ml kg −1 sample time −1 . No differences in milt production were observed among the three GnRHa doses. Histological evaluation of the testes indicated that, by day 21, control fish had entered into gonadal regression, containing almost exclusively spermatogonia. At the same time, testes from GnRHa-implanted fish still contained large numbers of spermatozoa in the testes, indicating that spermiation was still underway. Sperm density varied between 7.4 and 14.4×10 10 spermatozoa ml −1 , but remained unchanged in response to GnRHa implantation. Sperm motility was high at the beginning of the experiment (92–100%) and decreased significantly by day 27 (32–55%), whereas motility duration was unchanged (0.9–1.2 min). Neither sperm motility nor motility duration was affected by the GnRHa treatment. Finally, fertilization success was high (40–80%) and not significantly different in response to GnRHa implantation. These results demonstrate that GnRHa implants are effective at increasing milt production at the end of the spermiation period, maintaining high sperm density and not affecting, either positively or negatively, sperm quality and fertilization capacity.

Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.

Testis developments and puberty in the male Tokara (Japanese native) goat

In male Tokara (Japanese native) goats, testis development and the onset of puberty were studied histologically and observing sexual behaviour. Testes weight increased from 36±5.4(S.E.) g (n=5) at 3 months of age to 126±6.3g (n=6) at 12 months. The degree of the testis development differed among kids at 3 months of age and only one of four had produced spermatozoa in the testis. Large number of spermatozoa were always present in seminiferous tubules and epididymal ducts from 4 months of age. The diameter of seminiferous tubules increased from 133±9.9μm (n=4) at 3 months to 198±1.0μm (n=3) at 6 months with little increase thereafter. Mounting and pelvic thrusts onto a teaser doe started at from 9 to 14 weeks of age. Ejaculated semen contained sperm with good motility for the first time from 17 weeks. The male Tokara goat reaches puberty at around 4 months of age but testis development continues to 12 months.

Multicolor FISH Analysis of Chromosomal Breaks, Duplications, Deletions, and Numerical Abnormalities in the Sperm of Healthy Men

Transmitted de novo structural chromosomal abnormalities, the majority of which are paternally derived, can lead to abnormal reproductive outcomes as well as genetic diseases in offspring. We developed and validated a new multicolor FISH procedure (sperm ACM, which utilizes DNA probes specific for the alpha [1cen], classical, [1q12], and midi [1p36.3] satellites of chromosome 1) which utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical abnormalities plus two categories of structural aberrations: (1) duplications and deletions of 1pter and 1cen, and (2) chromosomal breaks within the 1cen-1q12 region. In healthy men, the average frequencies of sperm with duplications and deletions were (a) 4.5 +/- 0.5 and 4.1 +/- 1.3 per 10(4) involving 1pter and (b) 0.9 +/- 0.4 and 0.8 +/- 0.3 per 10(4) involving 1cen, respectively. The frequency of sperm exhibiting breaks within the 1cen-1q12 region was 14.1 +/- 1.2 per 10(4). Structural aberrations accounted for 71% of the abnormalities detected by sperm ACM, which was significantly higher than numerical abnormalities (P=2x10-8). Our findings also suggest that, for healthy men, (a) sperm carrying postmeiotic chromosomal breaks appear to be more prevalent than those carrying products of premeiotic or meiotic breakage or rearrangements, (b) the high frequency of chromosome breaks measured after "fertilization" by the hamster-egg cytogenetic method already appear to be present and detectable within human sperm by FISH, and (c) there are nonrandom and donor-specific distributions of breakpoint locations within 1q12 in sperm. FISH facilitates the analysis of much larger numbers of sperm than was possible when the hamster-egg method was used. Therefore, FISH-based procedures for simultaneously detecting chromosomal breaks, rearrangements, and numerical abnormalities in sperm may have widespread applications in human genetics, genetic toxicology, and reproductive medicine.

Sexing mammalian sperm for production of offspring: the state-of-the-art

Predetermination of sex in livestock offspring is in great demand and is of critical importance to providing for the most efficient production of the world's food supply. With the changes that have taken place in animal agriculture over the past generation the application of sex preselection to production systems becomes increasingly necessary. The current technology is based on the well-known difference in X- and Y-sperm in the amount of DNA present. The method has been validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content and embryo biopsy for sex determination. The technology incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation. It can also be used for intratubal insemination and for deep-uterine and conventional insemination in cattle. This semipractical sexing method, though currently impractical for some production systems (where large numbers of sperm are required for fertilization) could be used to provide a more flexible progeny-producing option in many livestock operations. Improvements in the production rate of sexed sperm continue as new technology is developed. High-speed sorting is one of the newer technological advances and is being used in our laboratory to increase sorted sperm throughput. With our original technology we sorted 350,000 sperm/h. We now sort 6 million of each sex, under routine conditions. Sorting only the X population results in about 18 million sperm/h. Improvements in the technology will no doubt lead to much greater usage of sexed sperm, depending on the species involved. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Solving the problems associated with inseminating low sperm numbers in the pig would be advantageous to the utilization of sexed sperm for some type of deep artificial insemination. Such a development would also enhance the economy of using lower sperm numbers with conventional artificial insemination (AI) and aid the swine industry worldwide. The use of sexed sperm for non-ordinary applications such as endangered species, laboratory animals, hobby or pet species is also of interest and will become a part of the move to be more reproductively efficient in the next millennium. Sexed sperm on demand over the next several years will provide livestock producers with many options in seeking to improve efficiency of production and improve quality of products to enhance consumer acceptability.

Postcopulatory sexual selection in Mediterranean fruit flies: advantages for large and protein-fed males

Previous laboratory studies of Mediterranean fruit flies, Ceratitis capitata (medflies), have identified large size and protein feeding as positive influences on the ability of males to secure copulations. In this study, we investigated whether large and protein-fed males experience additional advantages in terms of amount and distribution of sperm stored by mates. We also examined relationships between copula duration and sperm storage. Mates of large and protein-fed males were more likely to store sperm and to store more sperm than mates of small and protein-deprived males. Probability of sperm storage was associated with copula duration; all copulations lasting less than 100min failed whereas 98% lasting longer than 100min succeeded. Copulations involving sperm storage were longer if males were small or protein deprived or if the female was large, although there was no evidence of a relationship between copula duration and total sperm storage. Evidence from related studies suggests that variation in latency until sperm transfer, caused by size and diet, is a likely explanation for varying copula duration. Sperm tended to be stored asymmetrically between the female's two spermathecae, consistent with a mating system in which females maintain isolated populations of sperm from different males and later select between them. Storage was less asymmetric when large numbers of sperm were stored but there was little evidence that male size or diet affected this asymmetry. It is uncertain whether postcopulatory advantages of large and protein-fed male medflies arise from female preferences or male dominance through coercion or force.

BLADDER NECK COLLAGEN INJECTION RESTORES ANTEGRADE EJACULATION AFTER BLADDER NECK SURGERY

A 36-year-old man with a history of Y-V plasty of the bladder neck during childhood presented with primary infertility and low volume ejaculate. Physical examination was normal. Semen analyses consistently demonstrated a diminished ejaculate volume, as well as impaired sperm density and motility (see table). Post-ejaculate urine specimens contained large numbers of sperm, indicative of retrograde ejaculation. Initially, oral pseudoephedrine was prescribed for 4 weeks without any improvement in ejaculate volume. Subsequently, 2 cycles of intrauterine insemination using sperm recovered from bladder washes were unsuccessful. After a nonreactive skin test 7.5 cc collagen were injected submucosally and circumferentially at the bladder neck. Although initial postoperative semen analyses showed increased volumes and counts, the couple elected intrauterine insemination. Intrauterine insemination with an antegrade ejaculate 3 weeks postoperatively resulted in a pregnancy, which ended in a first trimester miscarriage. Collagen injection was repeated in identical fashion 6 months later because of decreasing semen volumes. Within 1 month postoperatively pregnancy was achieved using an antegrade ejaculate with intrauterine insemination. At the time of this report, gestation is progressing normally at 4 months. The patient had no difficulty with urination and examination of

Micromanipulation of gametes and embryos: Cryopreservation of a single human spermatozoon within an isolated zona pellucida.

There are several reasons for a need to cryostore a smallnumber of spermatozoa. Men who require testicular biopsybenefit tremendously if the presence of spermatozoa isknown before the onset of follicular recruitment in thepartner. This can usually be achieved by cryopreserving thebiopsy material or suspension of cells. But post-thawsuccess depends on the presence of relatively largenumbers of spermatozoa, since the addition of cryo-protectant requires removal by centrifugation whichdestroys many viable cells. The cut-off level forconventional freezing in mini-straws is 1200 spermatozoa,but many men have even less cells. Freezing of multiplesamples of very few spermatozoa would neverthelessguarantee several attempts at intracytoplasmic sperminjection (ICSI) and avoid repeated testicular aspirations incase further treatments are required.The recent of use of testicular spermatozoa has greatlyemphasized the need for the storage of aspirated testiculartissue or of spermatozoa that are recovered from suchtissue (Salzbrun et al ., 1996). Other methods of cryostoringsmall numbers of spermatozoa retrieved from the testisinclude the use of small droplets of medium which areplaced on a dry ice surface before freezing in liquidnitrogen, to give pregnancy rates similar to those obtainedwith fresh testicular spermatozoa (Gil-Salom et al ., 1996).Though both techniques are elegant and extremely useful,application falters when levels drop below one thousand.Special methods are necessary to store spermatozoa incases of extreme oligozoospermia. This circumstancearises constantly during ICSI when epididymal ortesticular spermatozoa are very rare. The most extremecases involve finding <100 spermatozoa, and sometimeswell below this number. This video shows a method ofovercoming this problem, by cryostoring single humanspermatozoa (Cohen et al ., 1997). It involves placing themin a zona pellucida after its oocyte has been removed. Thesequences in the video include the initial preparation ofzonae pellucidae, which came from oocytes that wereimmature when aspirated from their follicles, eggs whichdisplayed abnormal fertilization, and unfertilized mouseand hamster oocytes. Fine lesions were made in the zonaeusing mechanical methods. The ooplasm was thenaspirated from the oocytes and eggs, to provide emptyzonae for storage. The example shows a hamster profile.Up to 15 spermatozoa, are then isolated and injected intothe evacuated zona pellucida, which was crypopreservedusing glycerol; this cryopreservation procedure is notshown on this video. The final sequences illustrate therecovery of the frozen/thawed spermatozoa. In practice,only one zona pellucida among 50 was not recovered aftercryopreservation, and no spermatozoa were lost throughthe narrow incisions in the zona pellucida. Fertilizationrates using the cryopreserved spermatozoa for ICSI wereabove 65%.AcknowledgementsThis video was prepared with the assistance of Mina Alika-ni, Adrienne Reing, Toni Ferrarra, Sasha Sadowy and TimSchimmel.References

Some anatomical and behavioural aspects of reproduction in members of an unusual teleost family: the Phallostethidae

Male phallostethids have complex asymmetrical modifications of the pelvic girdle and associated structures, illustrated here for Neostethus bicornis and N. lankesteri. This is associated with modifications of the reproductive system in these and another species (Phenacostethus smithi): the atherinomorph-type testis leads into a duct system which is specialized for the secretion of a protein-rich seminal fluid. The spermatozoa are packaged as spermatophores in Neostethus spp., whereas any such organization is but transitory in P. smithi. Females produce relatively small numbers of large, filamented oocytes; large numbers of spermatozoa are present in the ovarian lumen, especially in neostethids. Mating behaviour of the two Neostethus spp. is described briefly on the basis of a small number of video recordings, and reconstructed with anaesthetized specimens: the male grasps the female, presumably using modified pelvic elements (the ctenactinia), this culminating in a bolus of spermatophores in a proteinaceous secretion being deposited in the vicinity of the female's oviducal opening. The sperm must somehow then migrate into the oviduct to fertilize the eggs prior to their being laid.

Ejaculate dynamics in butterflies: a strategy for maximizing fertilization success?

The butterfly spermatophore is a complex structure containing both sperm and accessory substances. Most of the sperm are anucleated ‘apyrene’ sperm, which do not fertilize the eggs. The fertilizing ‘eupyrene’ sperm comprise only 10-15 % of the total sperm number transferred to the female at mating. In addition to sperm, some butterfly species also provide the female with nutrients that are used for egg production and/or somatic maintenance. Many butterflies eclose with most resources available for reproduction already present. Consequently, resources have to be allocated to the different spermatophore components over consecutive matings. This study is the first to quantify eupyrene and apyrene sperm numbers in relation to spermatophore mass, mating history and larval diet. Spermatophore mass was significantly reduced on the male’s second mating. However, both eupyrene and apyrene sperm numbers were dramatically increased, as was the proportion of eupyrene sperm. Theory predicts that sperm numbers should increase under sperm competition. On males’ second matings, the probability of encountering virgin females is reduced. Males may therefore maximise fertilization success by providing these females with large numbers of sperm. Simultaneously, it may be advantageous for males to reduce their nutrient donation because of their lower paternity assurance. Larval diet affected mass of the first spermatophore but had no effect on sperm number. This suggests that males maintain sperm numbers when resources are limited, prioritising fertilization success over investment in offspring.

Effects of short-term exposure to hydroxyflutamide in utero on the development of the reproductive tract in male mice.

To determine if and when short-term ablation of androgen action compromises the development of the male reproductive tract in mice, the androgen receptor antagonist hydroxyflutamide was administered orally to pregnant FVB/N mice and the reproductive tracts of the male offspring were examined when adult. Hydroxyflutamide (30 mg per day) for 5 days from day 11 to day 15 of gestation caused hypospadias in all male progeny. However, testis weights, seminal vesicle weights, and serum testosterone levels were not affected (p > 0.05) but caput-corpus epididymal weights were 15% lower than controls (p < 0.02). Shorter periods of treatment that included day 14 or 15 caused hypospadias, but treatments that did not include days 14 and 15 did not (p < 0.002). Hydroxyflutamide (30 mg, once or twice daily for 2 consecutive days) between days 15 and 20 of gestation demonstrated that androgen ablation on days 15 & 16 caused hypospadias, absence of prostate, and scrotal location of the seminal vesicles with abdominal testes (p < 0.05). Males exposed later in pregnancy had prostates, but the weights were reduced (p < 0.001); testes were scrotal and seminal vesicles were abdominal; caput-corpus epididymal weights were 15-30% lower than controls (p < 0.05), but the tubule contained large numbers of spermatozoa. Furthermore, testis weights, serum testosterone, and the response of the testis to a human chorionic gonadotropin (hCG) challenge in vitro were not compromised by hydroxyflutamide, and seminiferous tubules exhibited normal spermatogenesis. When males that had been exposed to hydroxyflutamide on days 13 & 14, 15 & 16, 17 & 18 and 19 & 20 were housed with sexually mature females, pregnancies resulted only from the day 19 & 20 treatment group. Thus, there are long-term effects caused by short-term blockade of androgen action at critical times during pregnancy and such effects could result in the inability to impregnate, irrespective of any externally visible indications of developmental anomalies.

Localization of Spermatozoa and Leukocytes in Vagina and Uterovaginal Junction after Copulation in Japanese Quail(Coturnix coturnix japonica).

The aim of this study was to examine the mechanism by which the number of spermatozoa decreases in the oviduct of Japanese quail (Coturnix coturnix japonica) after copulation. We observed the vagina and uterovaginal junction (UVJ) histologically before and 0.5, 1, 3 and 6h after copulation. A large number of spermatozoa were observed in the lumen of vagina and UVJ 0.5h after copulation, followed by a markedly decreasing number in posterior vagina (pV) and UVJ 1h after copulation. Some of the spermatozoa in the vaginal and UVJ lumen showed an eosinophilic appearance. The number of leukocytes (polymorphonuclear leukocytes and lymphocytes) increased in the vaginal and UVJ lumen after copulation, and some of them were in close contact with spermatozoa. Spermatozoa were also localized in the mucosal epithelium and lamina propria just beneath the mucosal epithelium of UVJ 0.5h to 6h after copulation. These results suggest that the immunoreactions may be involved in the process of the decrease in the number of spermatozoa in the oviduct after copulation.

Electron microscopic study of human sperm membrane isolation

Our purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants. Donor semen samples collected after masturbation were stored at -70 degrees C and eventually pooled. Each attempt at sperm membrane isolation required 800 x 10(6) spermatozoa which were sonicated by ultrasound (40% output; Vibra Cell). The effect of sonication time (3 x 5, 3 x 15, and 180 sec) on membrane isolation was investigated. Sonicated samples were centrifuged (500 g, 5 min) and the supernatant was pipetted off. The supernatant of the centrifuged sample was layered on either a sucrose cushion (supernatant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifuged (100,000 g, 1 hr). Contents of supernatants of sonicated samples and fractions (sucrose interfaces) were then fixed in 1.0% tannic acid and 2.5% buffered glutaraldehyde and examined electron microscopically using standard procedures. (1) The optimal sonification time was found to be 3 x 15 sec. (2) Membrane isolation using a sucrose cushion was found to be inadequate, showing significant cellular contamination. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective. The advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.

Comparative studies of epididymal morphology and sperm distribution in dasyurid marsupials during the breeding season

The structural features of the epididymis and the number and distribution of spermatozoa along the duct, during the breeding season, were examined in two semelparous and three iteroparous dasyurid marsupials. Total numbers of epididymal spermatozoa were extremely low in all of these species when compared with epididymal sperm numbers in most other marsupials and eutherian mammals. Although semelparous dasyurids had significantly more epididymal spermatozoa than itcroparous species, very few spermatozoa were seen in the distal cauda epididymidis of any of the species examined. This coincided with distinct changes in duct shape and the surface area of the lumen in caudal regions which resulted in a reduced sperm storage capacity in the cauda epididymidis of these species. The data suggest that, like Antechinus stuartii (Taggart &amp; Temple‐Smith, 1990a), sperm content of the ejaculates in these species will be extremely low, and that sperm motility and/or transport in the female tract is highly efficient. The functional and evolutionary significance of the reproductive strategies of semelparous and iteroparous dasyurid marsupials is still obscure and further study is needed to determine if the length of sperm storage in the female and sperm competition for storage sites is related to sperm distribution in the male and mating activities. This study does, however, clearly indicate that large numbers of spermatozoa are not required to ensure successful fertilization in either semelparous or iteroparous members of the family Dasyuridae.

Can severe male factor infertility be treated without micromanipulation?

To present data on IVF in severe male factor infertility using modified IVF laboratory methods and to compare them with the reported data of gamete micromanipulation. Retrospective, not randomized. University of California, Irvine, Center for Reproductive Health. Seventy-one patients with severe oligoasthenozoospermia defined as total motile count in the pretreatment sample of < 5 x 10(6). Two groups were identified, group I with total motile count between 1.5 and 5 x 10(6) and group II with total motile count < 1.5 x 10(6). Treatment of the semen samples with mini-Percoll and the modification of standard IVF techniques. These modifications include insemination with larger numbers of sperm, the use of culture tubes for insemination of oocytes, and pooling oocytes together in one or more culture tubes. Fertilization and pregnancy rates (PRs). Additionally, the number of patients achieving cryopreservation and patients with previous failure to fertilize are specified. Eighty percent of the patients achieved fertilization with an overall rate of 38% per oocyte. Fifty-three ETs were performed and 23 clinical pregnancies (43% per transfer and 35% per cycle) were achieved. Fifty percent of the patients had excess embryos for cryopreservation. In group I the fertilization rate was 54% with a PR of 56% per transfer and 48% per cycle. In group II the fertilization rate was 25% with a PR per transfer of 32% and 24% per cycle. Of 28 patients who had previous failure of fertilization, 25 fertilized and 8 pregnancies were established. These data demonstrate that by the modification of standard laboratory methods for IVF, monospermic fertilization, cleavage, and a high clinical PR can be achieved in cases of severe male factor infertility without having to resort to micromanipulation.

Mechanism of chromosome elimination in the hybridogenetic spermatogenesis of allotriploid males between Japanese and European water frogs

Of 21 allotriploid males that possessed two genomes of Rana nigromaculata and one genome of Rana lessonae 10 produced a large number of spermatozoa in their testes. When 4 of these males were backcrossed with a female of R. nigromaculata, all of the resulting froglets were diploid in chromosome number and were completely R. nigromaculata type in appearance. These allotriploid males proved to have produced spermatozoa with one R. nigromaculata genome hybridogenetically. Therefore, their germ line cells were investigated for the mechanism of elimination of their R. lessonae chromosomes. In histological sections of testes, the great majority of spermatogonia (approximately 10(4) cells) between mitotic prometaphase and anaphase appeared normal in chromosome behavior, whereas 17 spermatogonia showed several chromosomes whose behavior deviated from the normal course during the same period. These deviant chromosomes concentrated together near the equatorial plate and remained stationary at anaphase. In metaphase chromosome preparations made from spermatogonia, 67 and 185 of the 477 chromosome spreads were diploid and triploid, respectively. The rest were aneuploid. Notably, 8 triploid spreads consisted of 26 or more normal chromosomes and 13 or fewer degenerate chromosomes. From these results it is concluded that a set of R. lessonae chromosomes is eliminated from some, but not all spermatogonia by becoming degenerate during the mitotic period.

Structure of the vaginal plugs generated by normal rats and by rats with partially removed seminal vesicles.

Normal male rats generate vaginal plugs that appear to be firmly apposed to the vagino-cervical junction and permit a large number of spermatozoa to reach the uterus. A few spermatozoa form entangled masses inside these plugs, as revealed by light microscopy. Males in which the seminal vesicles have been partially removed produce plugs that are smaller and softer than those generated by normal males, and the plugs display a cup-like structure at the proximal end. The cup-like structure is completely filled with spermatozoa that exhibit a characteristic arrangement in relation to the plug material. In this situation, the number of spermatozoa that reach the uterus is very much reduced. Experiments were also carried out to explore the restoration of sperm transport by addition of a vaginal plug. Such experiments involved successive matings of individual females with a seminal vesicle-deprived male and with a vasectomized male (which generated the plug) and also the intravaginal injection of seminal vesicle secretions after mating with a seminal vesicle-deprived male. In none of the experimental situations was transport of spermatozoa to the uterus restored, and the plug consisted of a large quantity of trapped spermatozoa inside a mass of coagulated proteins. The results suggest that the structure of the plug depends on the amount of seminal vesicle secretion present in the ejaculate and that the vaginal plug must be formed immediately after deposition of the sperm if spermatozoa are to reach the uterus.

Sperm-zona pellucida interaction in cynomolgus and rhesus macaques.

These experiments were carried out to establish and validate an in vitro system for studying macaque sperm-zona pellucida interaction. Sperm of rhesus and cynomolgus macaques were capacitated in vitro and incubated with cryopreserved zonae pellucidae. Homologous gamete incubations were tested, as well as cross-species combinations. Approximately 25% of macaque sperm bound to the zonae acrosome reacted within 1 minute of gamete coincubation, although the percentage of acrosome reactions in the sperm suspension was less than 1%. There was a small but consistent increase in the percent of acrosome reactions of zona sperm after an additional hour of incubation in sperm-free media. Similar results were obtained in the cross-species experiments, suggesting that zonae from the two macaque species can be used interchangeably in sperm-zona binding assays. Differences in the physiologic characteristics of the sperm of the macaque species were demonstrated. Cynomolgus sperm required activation with caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to bind to the zonae. Rhesus sperm were able to bind to the zonae and acrosome react in the absence of activators, although both sperm binding and percentage of acrosome reactions increased with the addition of activators. Large numbers of sperm from both macaque species bound to the zonae of hamster oocytes after treatment with activators, but the bound sperm did not acrosome react. These experiments demonstrate the importance of evaluating the acrosomal status of sperm when sperm-zona binding assays are performed with macaque gametes.

Reproduction of the temperate aspidochiroteStichopus mollis(Echinodermata: Holothuroidea) in New Zealand

Abstract Reproduction of the holothurian Stichopus mollis from the north-east coast of New Zealand was investigated through histological examination of the gonads. Changes in the gametogenic condition of the gonads are documented by the maturity index and oocyte size-frequency methods. The ovary or testis is located in the anterior coelom, and consists of two bilaterally symmetrical tufts of gonad tubules. S. mollis has an annual reproductive cycle, spawning in the austral summer. Males and females show synchronous development. Growth of the gonads begins in early spring, and involves the regrowth of the gonad basis, with subsequent branching and lengthening of the gonad tubules. In female sea cucumbers, oocytes develop from 5 µm to a maximum of 180 µm. Growth is characterised by the formation of larger oocytes surrounded by folJicular epithelium in the centre of the tubules, with a ring of smaller oocytes along the tubule wall. The testis retains a large spermatocyte layer throughout most of gonad growth. Few gonads were found to contain large numbers of spermatozoa. After spawning, the gonad tubules and basis are completely resorbed in both sexes so that no gonad tissue exists for 5–6 months. Observations from the South Island suggest that individuals do not undergo complete resorption, but maintain a resting phase gonad during the winter. Stichopus mollis, therefore, shows a different reproductive pattern with respect to latitude. Although the “tubule recruitment model” of Smiley (1988) may be representative of gonadal growth in S. mollis in southern New Zealand, in the warmer waters of north-east New Zealand the gonad is completely resorbed and the progressive recruitment of tubules cannot occur. These results emphasise the importance of location in studying reproduction in widely distributed holothurian species.

Transport and storage of spermatozoa in the female reproductive tract of the brown marsupial mouse, Antechinus stuartii (Dasyuridae)

Female brown marsupial mice were mated and changes in the number and distribution of spermatozoa were assessed in several regions of the reproductive tract at 1, 2, 3, 7, 10, 14 and 18 days after mating. Approximately 40 x 10(3) spermatozoa/side were present in the female reproductive tract between Days 1 and 7 after a single mating. This had decreased (to approximately 9 x 10(3) spermatozoa/side) by Days 10 and 14 after mating; by Day 18 no spermatozoa were recovered. The maximum number of spermatozoa recorded in a female tract was approximately 72 x 10(3) spermatozoa/side (Day 5 female, death in laboratory) and the minimum recorded was approximately 2 x 10(3) spermatozoa/side on Day 2 after mating. Between Days 1 and 7 after mating most spermatozoa were located in the uterus and lower isthmus (isthmus 1) and spermatozoa were rarely found in the lateral vaginae. By 24 h after mating most spermatozoa (approximately 60%) were found in isthmus 1, but approximately 35% were still present in the uterus. Histological observations of the lower isthmus at this time showed that large numbers of spermatozoa were present in both the lumen of the duct and the sperm storage crypts which are located in this region. By Day 7 after mating approximately 91% of all spermatozoa in the female tract were in isthmus 1, most of these being confined to the sperm storage crypts. On Days 10 and 14 after mating almost all spermatozoa in the tract were in the crypt regions of isthmus 1 and on Day 18 degenerating spermatozoa were observed. No special orientation or association of spermatozoa in relation to crypt cells was observed. These results show that, although the number of spermatozoa inseminated is low by mammalian standards sperm transport in this species is extremely efficient and a large proportion of spermatozoa reaches the isthmus before ovulation (approximately 1 in 1 to 1 in 7). Several observations may explain the remarkable success of these low numbers of spermatozoa, including specializations of the reproductive tract which may have a directing effect on sperm movement and the special relationship which exists between spermatozoa and the oviducal environment which results in viable sperm storage. Recent observations suggest that an unusual sinusoidal mode of progressive motility observed in this species, may also influence the success of the low numbers of ejaculated spermatozoa.