Predominance of genetic monogamy by females in a hammerhead shark, Sphyrna tiburo: Implications for shark conservation.

There is growing interest in the mating systems of sharks and their relatives (Class Chondrichthyes) because these ancient fishes occupy a key position in vertebrate phylogeny and are increasingly in need of conservation due to widespread overexploitation. Based on precious few genetic and field observational studies, current speculation is that polyandrous mating strategies and multiple paternity may be common in sharks as they are in most other vertebrates. Here, we test this hypothesis by examining the genetic mating system of the bonnethead shark, Sphyrna tiburo, using microsatellite DNA profiling of 22 litters (22 mothers, 188 embryos genotyped at four polymorphic loci) obtained from multiple locations along the west coast of Florida. Contrary to expectations based on the ability of female S. tiburo to store sperm, the social nature of this species and the 100% multiple paternity observed in two other coastal shark species, over 81% of sampled bonnethead females produced litters sired by a single male (i.e. genetic monogamy). When multiple paternity occurred in S. tiburo, there was an indication of increased incidence in larger mothers with bigger litters. Our data suggest that sharks may exhibit complex genetic mating systems with a high degree of interspecific variability, and as a result some species may be more susceptible to loss of genetic variation in the face of escalating fishing pressure. Based on these findings, we suggest that knowledge of elasmobranch mating systems should be an important component of conservation and management programmes for these heavily exploited species.

An algorithm to predict pregnancy in assisted conception

BACKGROUND: Male fertility potential cannot be measured by conventional parameters for assisted reproduction by intracytoplasmic sperm injection. This study determines the relationship between testicular and ejaculated sperm mitochondrial (mt) DNA deletions, nuclear (n) DNA fragmentation and fertilisation and pregnancy rates in ICSI. METHODS: Ejaculated sperm were obtained from 77 men and testicular sperm from 28 men with obstructive azoospermia undergoing ICSI. Testicular sperm were retrieved using a Trucut needle. MtDNA analysed using a long polymerase chain reaction. The alkaline Comet assay determined nDNA fragmentation. RESULTS: Of subjects who achieved a pregnancy (50%) using testicular sperm, only 26% had partners�??�?�¢?? sperm with wild type (WT) mtDNA. Of pregnant subjects (38%) using ejaculated sperm, only 8% had partner sperm with WT mtDNA.. In each, the successful group had less mtDNA deletions and less nDNA fragmentation. There were inverse relationships between pregnancy and mtDNA deletion numbers, size and nDNA fragmentation for both testicular and ejaculated sperm. No relationships were observed with fertilisation rates. An algorithm for the prediction of pregnancy is presented based on the quality of sperm nDNA and mtDNA. CONCLUSION: In both testicular and ejaculated sperm, mtDNA deletions and nDNA fragmentation are closely associated with pregnancy in ICSI.

The effects of short and long incubations on DNA fragmentation of testicular

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4 hr and 24 hr incubations, and after cryopreservation. The effect is intensified by post-thaw incubations. Testicular sperm to be used clinically in ICSI should be injected without delay.

Effects of vasectomy on spermatogenesis and fertility outcomes after assisted conception

Abstract BACKGROUND: Each year 40,000 men have a vasectomy in the UK whilst another 2400 request a reversal to begin a second family. Sperm can now be obtained by testicular biopsy and subsequently used in assisted conception with intracytoplasmic sperm injection (ICSI). The study aims were to compare sperm yields of men post-vasectomy or with obstructive azoospermia (OA) of unknown aetiology with fertile men and to assess any alteration in the clinical pregnancy rates after ICSI. METHODS: Testicular tissue was obtained by Trucut needle from men who had undergone a vasectomy >5yrs previously, had OA from other causes and from fertile men during vasectomy. Seminiferous tubules were milked to measure sperm yields. Numbers of Sertoli cells, spermatids and thickness of the seminiferous tubule walls were assessed using quantitative computerized analysis. RESULTS and CONCLUSIONS: Sperm yields/g testis were significantly decreased in men post-vasectomy and in men with OA, relative to fertile men. Significant reductions were also observed in early (40%) and mature (29%) spermatid numbers and an increase of 31% was seen in the seminiferous tubule wall (basal membrane and collagen thickness) of vasectomised men compared to fertile men. Clinical pregnancy rates in couples who had had a vasectomy were also significantly reduced.

Insulin Dependant Diabetes Mellitus: Implications for Male Reproductive Function.

BACKGROUND Diabetes mellitus (DM) is increasing in men of reproductive age. Despite this, the prevalence of diabetes in men attending fertility clinics is largely unknown. Furthermore, studies examining the effects of DM on sperm fertility potential have been limited to conventional semen analysis. METHODS Conventional semen analysis (semen volume, sperm count, motility and morphology) was performed for 27 diabetic (mean age 34 +/- 2 years) and 29 non-diabetic subjects (control group, men undergoing routine infertility investigations, mean age 33 +/- 1 years). Nuclear DNA (nDNA) fragmentation was assessed using the alkaline Comet assay and mitochondrial DNA (mtDNA) deletions by Long-PCR. RESULTS Other than a small, but significant, reduction in semen volume in diabetic men (2.6 versus 3.3 ml; P <0.05), conventional semen parameters did not differ significantly from control subjects. Diabetic subjects had significantly higher mean nDNA fragmentation (53 versus 32%; P <0.0001) and median number of mtDNA deletions (4 versus 3; P <0.05) compared with control subjects. CONCLUSIONS Diabetes is associated with increased sperm nuclear and mtDNA damage that may impair the reproductive capability of these men.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Differences In Metabolic Profile In the Testes of Streptozotocin Induced Diabetes Mellitus Mice As Detected By 1H NMR

Contrary to the traditional view, recent studies suggest that diabetes mellitus has an adverse influence on male reproductive function. Our aim was to determine the affect of diabetes on the testicular environment by identifying and then assessing perturbations in small molecule metabolites. Testes were obtained from control and streptozotocin induced diabetic C57BL/6 mice, two, four and eight weeks post treatment. Diabetic status was confirmed by HbA1c, non fasting blood glucose, physiological condition and body weight. Protein free, low molecular weight, water soluble extracts were assessed using 1H NMR spectroscopy. Principal Component Analysis of the derived profiles was used to classify any variations and specific metabolites were identified based on their spectral pattern. Characteristic metabolite profiles were identified for control and diabetic animals with the most distinctive being from mice with the greatest physical deterioration and loss of bodyweight. Eight streptozotocin treated animals did not develop diabetes and displayed profiles similar to controls. Diabetic mice had decreases in creatine, choline and carnitine and increases in lactate, alanine and myo-inositol. Betaine levels were found to be increased in the majority of diabetic mice but decreased in two animals with severe loss of body weight and physical condition. The association between perturbations in a number of small molecule metabolites known to be influential in sperm function, with diabetic status and physiological condition, adds further impetus to the proposal that diabetes influences important spermatogenic pathways and mechanisms in a subtle and previously unrecognised manner.

Virgin birth in a hammerhead shark.

Parthenogenesis has been documented in all major jawed vertebrate lineages except mammals and cartilaginous fishes (Class Chondrichthyes: sharks, batoids, chimeras). Reports of captive female sharks giving birth despite being held in the extended absence of males have generally been ascribed to prior matings coupled with long-term sperm storage by the females. Here we provide the first genetic evidence for chondrichthyan parthenogenesis, involving a hammerhead shark. This finding also broadens the known occurrence of a specific type of asexual development (automictic parthenogenesis) among vertebrates, extending recently raised concerns about the potential negative effect of this type of facultative parthenogenesis on the genetic diversity of threatened vertebrate species.

Advanced Glycation End Products (AGEs) accumulate in the Reproductive Tract of Men with Diabetes.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable affects of diabetes on sperm function. Specifically, a recent study has found significantly higher sperm nuclear DNA (nDNA) fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and 9 non-diabetic subjects. CML immunoreactivity was found through out the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells (cytoplasm and nuclei) of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggests that these compounds may play a hitherto unrecognized role in male infertility.

Assessment of fibronectin as a potential new clinical tool in andrology.

In this prospective clinical study, 892 patients with normal and impaired semen were examined in order to investigate the correlation between the concentration of fibronectin in seminal plasma and the motility of spermatozoa. The fibronectin concentration in seminal plasma, total sperm motility and linear sperm motility were measured. We report here a significant negative correlation between the fibronectin concentration in seminal plasma and total sperm motility (r=-0.3474). There was no link between varicocele and vasectomy, or between varicocele and variation in the concentration of fibronectin. It is concluded that higher concentrations of the acute-phase protein fibronectin may be a cause of severe reduction in sperm motility. The investigation of fibronectin concentrations in seminal fluid could be a new and helpful clinical tool in the assessment of male fertility.

Male Infertility: A Clinical Reflection

The introduction of intracytoplasmic sperm injection (ICSI) has led to an inappropriate decrease in interest in male fertility. It is apparent that light microscopy provides limited information and molecular techniques show that DNA abnormalities need to be considered further. Abnormalities include not only Yq11 deletions but also DNA strand breaks. Increases in advanced glycation end-products in sperm from well controlled diabetics may provide a mechanism for this damage in non-diabetics. In addition, much publicity is given to decreased male fertility: this is NOT confirmed as technical variations and differences in study populations make it difficult to draw conclusions. The generation of stem cell derived germ cells provides hope for men without germ cells but this is currently only experimental.

Necrospermia and chronic spinal cord injury.

OBJECTIVE: To determine whether improvement in quality of semen over 4 consecutive days of electroejaculation in men with chronic spinal cord injury (SCI) was consistent with epididymal necrospermia. DESIGN: Prospective study of a random sample of men with SCI. SETTING: A southeastern Australian SCI management center in collaboration with the specialist andrology service of a university-based department of obstetrics and gynecology in a tertiary referral hospital. PATIENT(S): Nine men with chronic spinal cord injury. INTERVENTION(S): Semen samples were obtained by using electroejaculation, and testicular biopsy samples were obtained by using fine-needle tissue aspiration. MAIN OUTCOME MEASURE(S): Semen analysis was performed according to World Health Organization criteria. Testicular biopsy and electron microscopy were done by using standard techniques. RESULT(S): During up to 4 days of consecutive-day electroejaculation, sperm motility and viability in semen obtained from men with chronic SCI increased by an average of 23% on days 2 and 3. The severity of the degenerative changes and the numbers of spermatozoa affected on day 1 became less marked by day 4. The changes were not present in late spermatids obtained from testicular biopsies. CONCLUSION(S): The asthenospermia of chronic SCI is similar to epididymal necrospermia and can be improved by consecutive-day electroejaculation.

Genetic disorders and spermatogenesis.

Male infertility affects one man in twenty and a genetic basis seems likely in at least 30% of those men. Genetic regulation of fertility involves the inter-related processes of testicular development, spermatogenesis (involving germ cell mitosis, meiosis and spermatid maturation), and their endocrine and paracrine regulation. In regard to spermatogenesis, particular attention has been given to the Yq11 region, where some spermatogenesis genes ('azoospermia factors') appear to be located. Several candidate genes have been identified but have not been shown to have a defined or essential role in spermatogenesis. Microdeletions of Yq11 are found in approximately 15% of azoospermic or severely oligospermic men. The complexity of the genetic control of male fertility is demonstrated by the evidence for genes involved in spermatogenesis and sexual differentiation on the X chromosome and autosomes. Better understanding of the genetic regulation of normal spermatogenesis will provide new probes for clinical studies; however, at present the majority of spermatogenic failure remains without an identified genetic linkage. The advent of intracytoplasmic sperm injection permits fertility in many previously sterile men and presents the possibility of their transmission of infertility; appropriate counselling is required.

Advanced glycation end products accumulate in the reproductive tract of men with diabetes

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility.

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 105 molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 105 molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.