Male Reproductive Abnormalities Research Articles

Antibiotic Therapy as Infertility Management

Today infertility is a common problem affecting as much as 15% of couples. Infertility is caused by a variety of reasons, with male reproductive system abnormalities accounting for a large percentage (approximately 35%) and the anatomical anomalies or lesions related to female reproductive organs equally responsible (also approximately 35%). Endometriosis, ovulation abnormalities, autoimmune disorders, genetic abnormalities and asymptomatic genital infections are also major causes of female and male infertility respectively. Unexplained infertility, presented in a large percentage of couples (25-30%), is usually due to undetectable microbial infections by the routine diagnostic techniques available. So far one of the most successful treatment methods available for unexplained infertility is the use of antimicrobial therapy. However, further research is required for the absolute confirmation of the benefits of the antimicrobial therapy as a means of infertility management.

Utility of sperm DNA fragmentation testing in different clinical scenarios of male reproductive abnormalities and its influence in natural and assisted reproduction.

One of the measurements of sperm quality that can be assessed with validated protocols is DNA fragmentation, the subject was profoundly dissected in the article by Agarwal et al. and that constitutes the backbone of this commentary (1).

A 4‐year study of the proportional distribution of male reproductive organ abnormalities in cattle slaughtered at Nyagatare abattoir, Eastern Rwanda

Male reproductive performance has more impact on overall herd productivity than in the female. In order to assess herd productivity in cattle in Nyagatare, Eastern Rwanda, the proportional distribution of male reproductive organ abnormalities was investigated in 3087 bulls slaughtered over a 4‐year period. The aim of the study was to investigate the proportional distribution of male reproductive organ abnormalities in exotic and indigenous bulls slaughtered over a 4‐year period at Nyagatare abattoir in Eastern Rwanda. Positioning of the testicles was observed and recorded as the animals were assembled before slaughter. After slaughter, the internal and external reproductive organs of all bulls were removed, labelled and analysed for pathology. Significantly more indigenous (79.01%) than exotic (20.99%) animals were slaughtered (P < 0.05; N = 3 087). Overall occurrence of abnormalities was significantly higher in exotic (20.83%) than in indigenous (10.33%) animals (P < 0.05). Overall, abnormal location of testicles was the most common abnormality (4.08%) followed by abnormal prepuce and penis (2.33%), orchitis (1.94%), enlarged vesicular gland (0.91%), scrotal hernia (0.87%), unilateral cryptorchidism (0.81%), testicular hypoplasia (0.81%) and hydrocoele (0.78%). Abnormal location of testicles, abnormal prepuce and penis, orchitis, testicular hypoplasia and enlarged vesicular glands had significantly higher prevalence in exotic animals than in indigenous animals (P < 0.05). There was no significant difference in the prevalence of unilateral cryptorchidism, hydrocoele and scrotal hernia between the exotic and indigenous breeds. The encountered reproductive abnormalities result in poor herd fertility manifesting as low first‐service conception rates, prolonged breeding seasons and reduced weaning weights accompanied by inevitable financial losses in beef production. The observed high proportion of male reproductive abnormalities in exotic breeds might offset the professed benefits from introduction of these exotic breeds with the aim of improving productivity.

Membrane Estrogen Receptor 1 is Required for Normal Reproduction in Male and Female Mice

Steroid hormones, acting through their cognate nuclear receptors, are critical for many reproductive and non-reproductive functions. Over the past two decades, it has become increasingly clear that in addition to cytoplasmic/nuclear steroid receptors that alter gene transcription when liganded, a small fraction of cellular steroid receptors are localized to the cell membranes, where they mediate rapid steroid hormone effects. 17β-Estradiol (E2), a key steroid hormone for both male and female reproduction, acts predominately through its main receptor, estrogen receptor 1 (ESR1). Most ESR1 is nuclear; however, 5-10 % of ESR1 is localized to the cell membrane after being palmitoylated at cysteine 451 in mice. This review discusses reproductive phenotypes of a newly-developed mouse model with a C451A point mutation that precludes membrane targeting of ESR1. This transgenic mouse, termed the nuclear-only ESR1 (NOER) mouse, shows extensive male and female reproductive abnormalities and infertility despite normally functional nuclear ESR1 (nESR1). These results provide the first in vivo evidence that membrane-initiated E2/ESR1 signaling is required for normal male and female reproductive functions and fertility. Signaling mechanisms for membrane ESR1 (mESR1), as well as how mESR1 works with nESR1 to mediate estrogen effects, are still being established. We discuss some possible mechanisms by which mESR1 might facilitate nESR1 signaling, as well as the emerging evidence that mESR1 might be a major mediator of epigenetic effects of estrogens, which are potentially linked to various adult-onset pathologies.

Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance.

Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs.

Transgenerational Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Stress Hormones and Behavior

Exposure to di-(2-ethylhexyl) phthalate (DEHP) has been linked to male reproductive abnormalities. Here, we assessed transgenerational actions of DEHP on several behaviors and stress responses. We used 2 doses of DEHP (150- and 200-mg/kg body weight) and a treatment regimen previously shown to produce transgenerational effects on male reproduction. Mice, 3 generations removed from DEHP exposure (F3), were tested for social behavior and anxiety on the elevated plus maze. We collected blood and pituitaries from undisturbed and restrained mice. Body weights, anogenital distances, and reproductive organ weights were collected at killing. In social interaction tests juvenile males from the DEHP lineage (200 mg/kg) displayed more digging and less self-grooming than did controls. Interestingly, 150-mg/kg lineage males, killed in early puberty, had smaller seminal vesicle weights than their controls. However, the 200-mg/kg males (killed on average 10 d later) did not show this effect. Females from a DEHP lineage had lower corticosterone concentrations than controls after restraint stress. We also found sex- and DEHP-specific mRNA expression changes in the pituitary in 2 of the 6 stress-related genes we measured. In particular, Gnas mRNA was elevated by the combination of DEHP lineage and stress. Thus, transgenerational effects of DEHP are noted in male behavior, and in females, DEHP had transgenerational effects on levels of corticosterone. Both of these results may be related to transgenerational modifications in the expression of several pituitary hormones involved in the hypothalamic-pituitary-adrenal axis.

Spermatogenic Alterations Induced by Organophosporus Compounds Profenofos, Chlorpyrifos and Synthetic Pyrethroid Lambada-cyhalothrin in Mice

Background: Fertility is declining in many countries and there has been substantial interest in the potential adverse effects of exposure to environmental hazardous chemicals, including pesticides on male reproduction, and it was evident that using pesticides play as the major reasons for sperm decline besides tested pesticides are used widely recently, that is why it was very important the investigate their draw back in fertilization. The objective of the present study focuses on the spermiotoxicity of some pesticides such as profenofos, chlorpyrifos, and lambda-cyhalothrin on male albino mice. Study Design: To assess the effect of tested pesticides on sperm morphology of male albino mice treated for 30, 60 and 90 consecutive days with different doses of pesticides (1/10, 1/40 and ADI LD50). Place and Duration of Study: Institute of animal health, Ministry of Agriculture, Egypt, between May 2011 and March 2012. Results: Data suggest a potential association between exposures to tested pesticides and decreased sperm quality and increased teratospermic (abnormal sperm morphology). Original Research Article Annual Research & Review in Biology, 4(6): 856-873, 2014 857 Further support for testicular toxicity comes from studies in laboratory albino mice that showed associations between exposure tested pesticides and sperm shape abnormalities, as well as dose-response relationships between exposure and a decline in epididymal sperm count and motility and increased abnormal sperm. Conclusion: Tested pesticides can cause male reproductive system abnormalities that include reduced sperm production. It is also possible that the genetic information of the sperm may potentially be altered prior to fertilization.

Fetal Growth Restriction Leads to Inhibition of Steroidogenesis.

Masculinization of the male reproductive tract occurs in humans from gestational weeks 10-22, and from gestational days 16-21 in rats. During this programming window, fetal testes produce a surge of testosterone causing reproductive masculinization. Disruption of this fetal testosterone production has been shown to result in various reproductive abnormalities including cryptorchidism and hypospadias. It is unknown what specifically causes these reproductive abnormalities aside from genetic defects of the steroidogenic pathway. In humans, in utero growth restriction is a well-known risk factor for cryptorchidism and hypospadias, but the mechanism is unknown. In addition, previous rat studies have suggested that fetal testosterone production can be disrupted by several factors including environmental exposure to anti-androgens such as dibutyl phthalate (DBP). In order to identify the mechanism underlying male reproductive malformation associated with in utero growth restriction, a maternal under-nutrition model was examined in the rat. Timed-pregnant Wistar rats were restricted to 50% (n=9) of the food eaten by ad libitum control dams (n=8) beginning on gestational day (GD) 3. Caloric restriction through GD17 resulted in a significant decrease in pup weight despite no significant decrease in litter size. Cultured fetal testes revealed a decrease in testosterone production of about 40% as indicated by radioimmunoassay. Additionally, mRNA levels of key components in the steroidogenic pathway including Scarb1 and Star were significantly decreased in the testis following in utero growth restriction. Analysis of postnatal endpoints following 50% caloric restriction (n=11) from GD3-18 indicated no evidence of undescended testes, hypospadias, and no significant decrease in anogenital distance at postnatal days 1, 14, or 28 as compared to ad libitum control animals (n=10). A coexposure study of in utero growth restriction and maternal dosing with the anti-androgen dibutyl phthalate was performed to determine if there was a cooperative effect on steroidogenesis. Analysis of fetal testes (GD17) following 50% maternal caloric restriction from GD3-17 (n=11) or exposure to DBP (250mg/kg bw/day) from GD15-17 without food restriction (n=7) showed a significant decrease in the expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star as compared to ad libitum controls (n=7). Moreover, when 50% caloric restriction was coupled with exposure to DBP (n=10), steroidogenic gene expression was further decreased. These results indicate that not only do in utero growth restriction and fetal exposure to DBP lead to a decrease in testosterone production separately, they also appear to work cooperatively to further disrupt the steroidogenic pathway. This interaction between chemical endocrine disruptor exposure and in utero growth restriction could begin to explain the mechanisms associated with human male reproductive abnormalities.

Differential expression of ATF3 in male rats with hypospadias induced by combination of Di-n-butyl phthalate and glucocorticoids

Objective To verify the differential expression of ATF3 in the genital tubercle (GT) of rats with hypospadias induced by maternal exposure to Di-n-butyl phthalate (DBP) or DBP + dexamethasone (DXM) in order to further explore the mechanism of hypospadias. Methods Forty pregnant SD rats were randomly divided into four groups which were given: group A, soybean oil oral gavage 2 ml/day;DBP (700 mg/kg gavage) + DXM (0. 1 mg/kg sc-injection) during gestational day (GD) 14-18. Pregnant rats were killed at GD 19, birth weight (BW) and anogenital distance (AGD) were recorded in the fetal rats, GT was harvested to verify the expression of ATF3 by Western blotting and immunohistochemistry. Results DXM alone had no effect except to reduce BW but could increase the teratogenic effect of DBP when combined with DBO. The BW and AGD/BW in group D were significantly lower than in group C. The expression of ATF3 had statisticall significant difference between groups C or D and group A, as well as between groups C and D (P ＜ 0. 05). ATF3 was mainly located in the urethral epithelium and mesenchyme. Conclusion These results suggests that exposure to common environmental chemicals in combination with environmental endocrine disrupters may increase the risk of male reproductive abnormalities,such as hypospadias. Up-regulation of ATF3 may affect urethral fold fusion, and then lead to the occurrence of hypospadias. Key words: ATF3; Glucocorticoids; Di-n-butyl phthalate; Hypospadias

Vinclozolin Exposure in Utero Induces Postpubertal Prostatitis and Reduces Sperm Production via a Reversible Hormone-Regulated Mechanism

Vinclozolin is an endocrine-disrupting chemical (EDC) that binds with high affinity to the androgen receptor (AR) and blocks the action of gonadal hormones on male reproductive organs. An alternative mechanism of action of Vinclozolin involves transgenerational effects on the male reproductive tract. We previously reported in utero Vinclozolin exposure-induced prostatitis (prostate inflammation) in postpubertal rats concurrent with down-regulation of AR and increased nuclear factor-kappaB activation. We postulated the male reproductive abnormalities induced by in utero Vinclozolin exposure could be reversed by testosterone supplementation, in contrast to the permanent modifications involving DNA methyltransferases (Dnmts) described by others. To test this hypothesis, we administered high-dose testosterone at puberty to Vinclozolin-treated rats and determined the effect on anogenital distance (AGD); testicular germ cell apoptosis, concentration of elongated spermatids, and the onset of prostatitis. Concurrently we examined Dnmt1, -3A, -3B, and -3L mRNA expression. Consistent with previous reports, in utero exposure to Vinclozolin significantly reduced AGD, increased testicular germ cell apoptosis 3-fold, reduced elongated spermatid number by 40%, and induced postpubertal prostatitis in 100% of exposed males. Administration of high-dose testosterone (25 mg/kg) at puberty normalized AGD, reduced germ cell apoptosis, and restored elongated spermatid number. Testosterone restored AR and nuclear factor-kappaB expression in the prostate and abolished Vinclozolin-induced prostatitis. Altered Dnmt expression was evident with in utero Vinclozolin exposure and was not normalized after testosterone treatment. These data demonstrate in utero Vinclozolin-induced male reproductive tract abnormalities are AR mediated and reversible and involve a mechanism independent of Dnmt expression.

Glucocorticoids Amplify Dibutyl Phthalate-Induced Disruption of Testosterone Production and Male Reproductive Development

Common male reproductive abnormalities including cryptorchidism, hypospadias, and low sperm counts may comprise a testicular dysgenesis syndrome (TDS), resulting from fetal testis dysfunction during a critical developmental period involving reduced androgen production/action. The recent increase in TDS prevalence suggests environmental/lifestyle factors may be etiologically important. The developing fetus is exposed to multimodal challenges, and we hypothesized that exposure to a combination of factors rather than single agents may be important in the pathogenesis of TDS. We experimentally induced fetal testis dysfunction in rats via treatment of pregnant females daily from embryonic day (e) 13.5 to e21.5 with vehicle, 100 or 500 mg/kg . d dibutyl phthalate (DBP), 0.1 mg/kg . d dexamethasone (Dex), or a combination of DBP + Dex. In adulthood, penile length/normality, testis weight/descent, prostate weight, and plasma testosterone levels were measured plus anogenital distance (AGD) as a measure of androgen action within the masculinization programming window. Intratesticular testosterone and steroidogenic enzyme gene expression were measured in fetal testes at e17.5. High-dose DBP reduced fetal intratesticular testosterone and steroidogenic gene expression; induced mild hypospadias (31%) and cryptorchidism (53%); and reduced penile length, AGD, and testis and prostate weight in adulthood. Dex alone had no effect except to reduce birth weight but amplified the adverse effects of 500 mg/kg . d DBP and exacerbated the effects of 100 mg/kg . d DBP. All adverse effects were highly correlated to AGD, emphasizing the etiological importance of the masculinization programming window. These findings suggest that exposure to common environmental chemicals in combination with, for example, maternal stress, may increase the risk of common male reproductive abnormalities, with implications for human populations.

Recovery of suppressed male reproduction in mice exposed to progesterone during embryonic development by testosterone

The present study aimed to examine whether transplacental exposure to progesterone caused male reproductive abnormalities and whether the changes can be reversed after testosterone administration. Progesterone was injected to mice on day 1, 3, and 7 of pregnancy. The male pups (F1 generation) were allowed to grow for 50 days and assessed for reproductive performance. Gestational exposure to progesterone (7 mg/kg body weight) resulted in significant body weight gain with a decrease in reproductive tissue indices in mice. Total sperm count, viable sperm, and motile sperm decreased in experimental mice. Hypo-osmotic swelling test revealed that experimental mice sperm membrane integrity was severely altered. The activity levels of testicular steroidogenic marker enzymes (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B) and hydroxysteroid (17-beta) dehydrogenase 1 (HSD17B)) decreased significantly in mice exposed to progesterone during embryonic development when compared with the controls. The levels of serum testosterone decreased with an increase in serum FSH and LH in mice exposed to progesterone during embryonic development. Prenatal exposure to progesterone caused significant reduction in the number of spermatozoa and increase in the lumen of seminiferous tubule. The experimental mice that cohabited with normal females showed fertility reduction. Administration of testosterone (4.16 mg/kg body weight) on postnatal day 20, 30, and 40 to progesterone-exposed prenates resulted in recovery of progesterone-induced suppressed male reproduction. It is suggested that the impairment of male reproduction in mice exposed to progesterone during embryonic development could be mediated through the inhibition of testosterone production. These results also indicate that in utero exposure to progesterone affects male reproduction and that supplementation of testosterone restores the suppressed male reproduction.

Distinct expression of three estrogen receptors in response to bisphenol A and nonylphenol in male Nile tilapias (Oreochromis niloticus)

Environmental estrogens, such as bisphenol A (BisA) and nonylphenol (NP), have been shown to affect the estrogen receptor (ER) expression and induce male reproductive abnormalities. To elucidate molecular mechanisms of action of xenoestrogenic chemicals on the expression of estrogen receptors in the testes of Nile tilapia (Oreochromis niloticus), three full-length cDNAs respectively encoding ntERalpha, ntERbeta1 and ntERbeta2 were cloned from testes. The amino acid sequences of ntERalpha, ntERbeta1 and ntERbeta2 showed a high degree of similarity to the relevant fish species. Tissue-specific expression study showed that three receptors were highly expressed in pituitary, liver, testis, kidney and intestine tissues. The ntERalpha, ntERbeta1 and ntERbeta2 mRNA expressions were significantly higher at the sexual early recrudescing stage than at other recrudesced stages. After being exposed to xenoestrogens from weeks 2 to 4, the ntERalpha mRNA levels were increased significantly in testes after NP treatment at all sampling times or after 4 weeks of exposure to BPA. The ntERbeta1 mRNA levels remained unchanged, while a significant decrease of the ntERbeta2 mRNA level was observed in testes after exposure to NP and BPA. The present study demonstrates that the regulation of all three ntER subtypes in testes may act via different molecular mechanisms of exposure to NP and BPA.

Ontogenèse et régulations des fonctions testiculaires chez le fœtus humain

Two major functions are assumed by the testis: the production of male gametes (that is, spermatozoa) and the production of steroid hormones. Both two functions are established during fetal life and are essential to the adult fertility and the masculinization of the internal tract and genitalia. For many years, our laboratory has been interested in the ontogeny of those two functions in rodents and, since 2003, in collaboration with gynecology and obstetrics service of professor R. Frydman in Antoine-Béclère hospital, we have studied them in human. The first aim of this work was to improve the global knowledge of the human fetal testis development by using both our experimental data and the literature. Then, we focused on the different defects that can occur during the fetal testis development. Indeed, male reproductive abnormalities have been steadily increasing since the last decades and are thought to be related to the concomitant increase of the concentration of contaminants and particularly of endocrine disruptors in the environment. Thus, we decided to study the effect of endocrine disruptors on human fetal testis and, more particularly, the effect of phthalates, by using an organ culture system developed for human. In contrast to the data obtained in rat, mono (ethylhexyl)-phthalate (MEHP), an active metabolite of the most widespread phthalate in the environment, does not disturb the steroidogenic function. On the other hand, it has a negative effect on the male germ cells number. This study is the first experimental demonstration of a negative effect of phthalates directly on human fetal testis.

Testing new dairy cattle for disease can boost herd health, cut costs

Dairy producers seldom test or examine incoming cattle, although these important biosecurity practices are recommended. This pilot project examined risk management decisions that producers make when faced with test-positive animals in purchased groups of dairy cattle, in order to provide information on disease risks and conditions that could affect animal health and performance. New arrivals to seven herds at dairy farms in four California counties were examined and tested for a range of conditions. The most common findings were bovine leukosis virus (33% of cattle purchased) and male reproductive abnormalities (16% of bulls purchased). Once testing results were known, producers made a variety of risk management decisions. Although testing costs for some conditions outweigh the benefits of finding an infected animal, an individual producer's decision to test new animals most likely depends on their knowledge of the pros and cons as well as their risk tolerance.

Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities

We have recently suggested that homozygosity for a specific 'AGATA' haplotype within a approximately 50 kb linkage disequilibrium (LD) block of the gene for estrogen receptor alpha (ESR1) may raise the susceptibility to cryptorchidism by enhancing estrogenic effects of environmental endocrine disruptors (EEDs). Haplotype analysis of ESR1 was performed in 328 Japanese subjects, i.e. 70 patients with micropenis (MP), 43 patients with hypospadias (HS), 80 patients with spermatogenic failure (SF) and 135 control males. Genotyping was performed by the 5' nuclease assay. The LD block was identified in each of the patient groups and in the control males. The frequency of homozygotes for the specific 'AGATA' haplotype was markedly higher in the HS patients [P = 0.0000033, odds ratio [OR] = 11.26] and slightly higher in the MP patients (P = 0.034, OR = 3.64) than in the control males, and the 'AGATA' haplotype was strongly associated with HS (P = 0.0000022, OR = 11.26) and weakly associated with MP (P = 0.040, OR = 3.64) in a recessive mode. There was no significant difference between the SF patients and the control males. Our results support the hypothesis that homozygosity for the specific ESR1 'AGATA' haplotype may increase the susceptibility to the development of male genital abnormalities in response to estrogenic EEDs.

Perspectives: The Possible Influence of Assisted Reproductive Technologies on Transgenerational Reproductive Effects of Environmental Endocrine Disruptors

Demasculinization by environmental endocrine-disrupting chemicals (EDCs) is observed in many animal species but less evident in humans. Rodent studies with gestational exposure to either the fungicide vinclozolin or the insecticide methoxychlor demonstrate impaired male fertility with abnormal DNA methylation patterns in spermatozoa. Once established, these epigenetic changes may be permanent and thus paternally passed to subsequent generations. Conclusive evidence of a similar phenomenon in humans has not been established, but several observations bring up the possibility. Some, but not all, studies show an increase in male genital abnormalities after prenatal EDC exposure. Other studies demonstrate sperm abnormalities in males with EDC contact, although it is unclear as to whether this is due to prenatal or postnatal exposure. Although not examined in males with EDC exposure, one study shows gamete DNA methylation abnormalities in males with severe oligospermia. A subsequent study failed to corroborate these findings. The use of assisted reproductive techniques including intracytoplasmic sperm injection has removed natural selection barriers thus enabling reproduction in males that would otherwise be sterile. This review explores the hypothesis that prenatal EDC exposure results in transgenerational male reproductive abnormalities propagated by the use of assisted reproductive technologies.

Low concentrations mono-butyl phthalate stimulates steroidogenesis by facilitating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1)

Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is widely distributed environmental contaminant. Although previous studies have demonstrated that DBP led to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists, DBP and its active metabolite, mono-butyl phthalate (MBP), have been demonstrated no affinity for the androgen receptor, but rather exert anti-androgenic effect by altering testosterone biosynthesis. Furthermore, all these results were obtained from very high administrations of DBP or MBP. The purpose of this study was to determine the onset and the site of action of relatively low concentration of MBP on steroidogenesis in vitro. The mouse Leydig tumor cells (MLTC-1) was employed as a cellular model to investigate the effect of MBP on steroidogenesis. Various concentrations of MBP (1, 10, 100 and 1000nmol/l) and its solvent dimethyl sulfoxide (DMSO) were added to the medium for 24h followed by stimulation of some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), forskolin, cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC) and pregnenolone. Progesterone in the medium and amounts of intracellular cAMP were measured by RIA. Expression of steroidogenic acute regulatory protein (StAR) was monitored by real-time PCR and Western blotting. The results revealed that the increases of progesterone production in the presence of hCG, CT, forskolin and 8-Br-cAMP were augmented by MBP. In contrast, the levels of intracellular cAMP exhibited no statistical significance when MLTC-1 cells were treated as above. These results implied that the site in the steroid biosynthesis pathway affected by MBP occurs after PKA activation in MLTC-1 cells. Moreover, supplementing the medium with 22R-HC and pregnenolone as progesterone precursors for P450 side chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), respectively, resulted in no rise in progesterone production, making clear that MBP did not influence the P450scc and 3beta-HSD but on the rate-limiting step, cholesterol transportation into mitochondria. In fact, the above results were confirmed by the upgraded StAR expression in MBP-treated cells. These data support that MBP promotes steroid hormone production by facilitating StAR expression in MLTC-1 cells.

Further Characterization of Reproductive Abnormalities in mCd59b Knockout Mice: A Potential New Function of mCd59 in Male Reproduction

CD59 is a GPI-linked membrane protein that inhibits formation of the membrane attack complex of complement. We reported recently that mice have two CD59 genes (termed mCd59a and mCd59b), and that the targeted deletion of mCd59b (mCd59b-/-) results in spontaneous hemolytic anemia and progressive loss of male fertility. Further studies of the reproductive abnormalities in mCd59b-/- mice reported in this study revealed the presence of abnormal multinucleated cells and increased apoptotic cells within the walls of the seminiferous tubules, and a decrease in the number, motility, and viability of sperm associated with a significant increase in abnormal sperm morphologies. Both the capacitation-associated tyrosine phosphorylation and the ionophore-induced acrosome reaction as well as luteinizing hormone, follicle-stimulating hormone, and testosterone serum levels were similar in mCd59b-/- and mCd59b+/+. Surprisingly, the functional deficiency of the complement protein C3 did not rescue the abnormal reproductive phenotype of mCd59b-/-, although it was efficient in rescuing their hemolytic anemia. These results indicate that the male reproductive abnormalities in mCd59b-/- are complement-independent, and that mCd59 may have a novel function in spermatogenesis that is most likely unrelated to its function as an inhibitor of membrane attack complex formation.

Dietary oestrogens and male fertility potential

Reports of increased incidences of male reproductive abnormalities and falling sperm counts have prompted interest into the nature of these threats to global fertility. Xenoestrogens have been flagged as major culprits but to date, little is known about the effects of dietary phytoestrogens on male reproductive health. These non-steroidal oestrogens of plant origin are potent endocrine disruptors that modulate normal physiological functions. Phytoestrogens have become a major component in the typical Western fast food diet over the last few decades. Soy formula milk is another common source of phytoestrogens, now used increasingly as an alternative to breast or cow's milk for infants with allergies. This use is of particular concern since the most vulnerable periods for oestrogenic insult are thought to be the pre- and neonatal periods when irreversible damage can be inflicted on the developing germinal epithelium. Studies into the safety of phytoestrogens are urgently needed either to allay fears or increase awareness of the effects of our modern diet on future fertility.