Testicular Daily Sperm Production Research Articles

Exome-informed formulations of food proteins enhance body growth and feed conversion efficiency in ad libitum-fed mice

Meeting requirements for dietary proteins, especially of essential amino acids (EAAs), is critical for the life-long health of living organisms. However, defining EAA targets for preparing biologically-matched nutrition that satisfies metabolic requirements for protein remains challenging. Previous research has shown the advantages of ‘exome matching’ in representing the specific requirement of dietary AAs, where the target dietary AA profile was derived from in silico translation of the genome of an organism, specifically responsible for protein expression (the ‘exome’). However, past studies have assessed these effects in only one sex, for few parameters (body mass and composition), and have used purified diets in which protein is supplied as a mixture of individual AAs. Here, for the first time, we utilise a computational method to guide the formulation of custom protein blends and test if exome matching can be achieved at the intact protein level, through blending standard protein ingredients, ultimately leading to optimal growth, longevity and reproductive function. Mice were provided ad libitum (ad lib) access to one of the four iso-energetic protein-limited diets, two matched and two mis-matched to the mouse exome target, and fed at a fixed protein energy level of 6.2%. During or following 13-weeks of feeding, the food intake, body growth, composition and reproductive functions were measured. Compared to the two mis-matched diets, male and female animals on the exome-matched diet with protein digestibility correction applied, exhibited significantly improved growth rates and final body mass. The feed conversion efficiency in the same diet was also increased by 62% and 40% over the worst diets for males and females, respectively. Male, not female, exhibited higher accretion of lean body mass with the matched, digestibility-corrected diet. All reproductive function measures in both sexes were comparable among diets, with the exception of testicular daily sperm production in males, which was higher in the two matched diets versus the mis-matched diets. The results collectively demonstrate the pronounced advantages of exome-matching in supporting body growth and improving feed conversion efficiency in both sexes. However, the potential impact of this approach in enhancing fertility needs further investigation.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Integrated Analyses of Phenotype and Quantitative Proteome of CMTM4 Deficient Mice Reveal Its Association with Male Fertility

The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family that has been implicated in male reproduction. CMTM4 is an evolutionarily conserved member that is highly expressed in the testis. However, its function in male fertility remains unknown. Here, we demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Using Western blotting and immunohistochemical analyses, we found CMTM4 expression to be decreased in poor-quality human spermatozoa, old human testes, and testicular biopsies with nonobstructive azoospermia. Using CRISPR-Cas9 technology, we knocked out the Cmtm4 gene in mice. These Cmtm4 knockout (KO) mice showed reduced testicular daily sperm production, lower epididymal sperm motility and increased proportion of abnormally backward-curved sperm heads and bent sperm midpieces. These mice also had an evident sub-fertile phenotype, characterized by low pregnancy rates on prolonged breeding with wild type female mice, reduced in vitro fertilization efficiency and a reduced percentage of acrosome reactions. We then performed quantitative proteomic analysis of the testes, where we identified 139 proteins to be downregulated in Cmtm4-KO mice, 100 (71.9%) of which were related to sperm motility and acrosome reaction. The same proteomic analysis was performed on sperm, where we identified 3588 proteins with 409 being differentially regulated in Cmtm4-KO mice. Our enrichment analysis showed that upregulated proteins were enriched with nucleosomal DNA binding functions and the downregulated proteins were enriched with actin binding functions. These findings elucidate the roles of CMTM4 in male fertility and demonstrates its potential as a promising molecular candidate for sperm quality assessment and the diagnosis or treatment of male infertility.

Profertility effects of Shilajit on cadmium-induced infertility in male mice.

Shilajit is claimed as a Vajikarak (aphrodisiac) and used for the treatment of male infertility by traditional healers of the Indian subcontinent. Therefore, the present investigation was designed to assess the effectiveness of Shilajit for treatment of male infertility resulting from exposure to perilous chemicals. Effect of daily oral administration (p.o.) of Shilajit (50mg, 100mg and 200mg/Kg BW) was investigated for a single spermatogenic cycle (35days) in cadmium-induced (2mg/Kg BW, p.o. for 35days) infertile adult (12-14week) swiss male mice. Shilajit treatment increased weights of reproductive organs, testicular daily sperm production, activities of testicular Δ5 3β-HSD and 17 β-HSD enzymes and serum level of testosterone. Histopathological evaluation of testis revealed that Shilajit restored spermatogenesis as reflected by a gradual augmentation in germ cell layers with increased doses of Shilajit compared to cadmium-treated mice. Further, Shilajit treatment reverted back the adverse effects of cadmium on motility and concentration of spermatozoa. Secretory activities of the epididymis and seminal vesicle and libido, fertility and the number of litters per female were also improved by Shilajit in cadmium-treated mice. Results thus suggest the potent androgenic nature of Shilajit and its role in fertility improvement against cadmium-induced infertility.

Alleviative effect of resveratrol on polyvinyl chloride-induced reproductive toxicity in male Wistar rats

The present study was aimed to investigate the effect of PVC on reproductive competence in adult male Wistar rats. Further, the study also encompasses the protective effect of trans-resveratrol on PVC-induced reproductive toxicity in rats. Adult male rats weighing 210–240 g were administered with either PVC at two different doses 100 and 500 mg/kg body weight, orally, daily for 60 days or resveratrol (20 mg/kg body weight/day) through gavage for 60 days on alternate days or both PVC (500 mg/kg body weight) and resveratrol. The results revealed significant reduction in the weights of reproductive organs, epididymal sperm count, viable-, motile-, and HOS-tail coiled sperm and testicular daily sperm production, steroidogenic enzyme activities, serum testosterone levels in PVC treated rats. Conversely the levels of lipid peroxidation increased significantly with a decrease in activity levels of antioxidant enzymes in the testis of PVC exposed rats. Exposure to PVC resulted in reduction in epithelial thickness and seminiferous tubule diameter. No significant changes in the selected reproductive variables were observed in the resveratrol alone treated control rats, whereas, co-administration of resveratrol and PVC resulted in a significant improvement in steroidogenesis and spermatogenesis and mitigated oxidative stress over PVC exposed rats.

The protective effects of zinc in lead-induced testicular and epididymal toxicity in Wistar rats.

The aim of this study was to investigate the beneficial effects of zinc (Zn) in preventing lead (Pb)-induced reproductive toxicity in Wistar rats. The rats were divided into four groups, namely, control group, Pb group, Zn group, and Pb + Zn group. Animals were exposed to Pb (819 mg of Pb/L) or Zn (71 mg of Zn/L) or both through drinking water for 65 days. Rats exposed to Pb showed decreased weights of testes and accessory sex organs. Significant decrease in the testicular daily sperm production, epididymal sperm count, motility, viability, and number of hypoosmotic tail coiled sperm was observed in Pb-exposed rats. Testicular 3β- and 17β-hydroxysteroid dehydrogenase activity levels and circulatory testosterone levels were also decreased significantly in Pb-exposed rats. A significant increase in the lipid peroxidation products with a significant decrease in the activities of catalase and superoxide dismutase were observed in the testes and epididymis of Pb-exposed rats. Moreover, the testicular architecture showed lumens devoid of sperm in Pb-exposed rats. Supplementation of Zn mitigated Pb-induced oxidative stress and restored the spermatogenesis and steroidogenesis in Pb-exposed rats. In conclusion, cotreatment of Zn is effective for recovering suppressed spermatogenesis, steroidogenesis, elevated oxidative status, and histological damage in the testis of rats treated with Pb.

Prenatal exposure to aflatoxin B1: developmental, behavioral, and reproductive alterations in male rats.

Previous studies have shown that aflatoxin B1 (AfB1) inhibits androgen biosynthesis as a result of its ability to form a high-affinity complex with the steroidogenic acute regulatory protein. The results of the present study demonstrate the postnatal effects of in utero exposure to AfB1 in the rat. Pregnant Wistar rats were given 10, 20, or 50 μg AfB1/kg body weight daily from gestation day (GD) 12 to GD 19. At parturition, newborns were observed for clinical signs and survival. All animals were born alive and initially appeared to be active. Male pups from control and AfB1-exposed animals were weaned and maintained up to postnatal day (PD) 100. Litter size, birth weight, sex ratio, survival rate, and crown-rump length of the pups were significantly decreased in AfB1-exposed rats when compared to controls. Elapsed time (days) for testes to descend into the scrotal sac was significantly delayed in experimental pups when compared to control pups. Behavioral observations such as cliff avoidance, negative geotaxis, surface rightening activity, ascending wire mesh, open field behavior, and exploratory and locomotory activities were significantly impaired in experimental pups. Body weights and the indices of testis, cauda epididymis, prostate, seminal vesicles, and liver were significantly reduced on PD 100 in male rats exposed to AfB1 during embryonic development when compared with controls. Significant reduction in the testicular daily sperm production, epididymal sperm count, and number of viable, motile, and hypo-osmotic tail coiled sperm was observed in experimental rats. The levels of serum testosterone and activity levels of testicular hydroxysteroid dehydrogenases were significantly decreased in a dose-dependent manner with a significant increase in the serum follicle-stimulating hormone and luteinizing hormone in experimental rats. Deterioration in the testicular and cauda epididymal architecture was observed in experimental rats. The results of fertility studies revealed a significant decrease in the mating index in experimental rats with an increase in the pre- and post-implantation losses in rats mated with prenatal AfB1-exposed males, indicating poor male reproductive performance. These results indicate that in utero exposure to AfB1 severely compromised postnatal development of neonatal rats, and caused a delay in testes descent and reduction in steroidogenesis and spermatogenesis that were accomplished by suppressed reproduction at adulthood.

Neonatal exposure to benzo[a]pyrene decreases the levels of serum testosterone and histone H3K14 acetylation of the StAR promoter in the testes of SD rats

Although benzo[a]pyrene (BaP) is an environmental endocrine disrupter, it has been unclear whether neonatal exposure to BaP affects the testosterone level and, if so, whether this influence persists into adulthood. In this present study, we gave neonatal rats (through oral gavages) doses of 0, 5, 10, or 25mg/kgday of BaP in corn oil from postnatal day 1 (PND 1) to PND 7. The rats were sacrificed at PND 8, PND 35, and PND 90. BaP exposure was confirmed through the induction of liver and testis CYP1A1 mRNA expression at PND 8 (i.e., immediately after exposure). The testicular daily sperm production and the sperm counts of the epididymis cauda at PND 90 were significantly lower than those of the control. The serum testosterone levels decreased markedly at PND 8, PND 35, and PND 90 after neonatal BaP exposure relative to those of the control. The mRNA expressions of StAR also decreased relative to those of the control at PND 8, PND 35, and PND 90, although the mRNA expressions of P450c17 and 17β-HSD were suppressed significantly only at PND 8. To further elucidate the mechanism of the persistent decrease in the mRNA expression of StAR, we determined the histone acetylation level in the StAR promoter. The extent of acetylation of H3K14 in the determined region decreased after neonatal exposure to BaP; this phenomenon persisted to the adult stage. Our results indicate that neonatal exposure to BaP damages testosterone production and sperm counts in the long term, possibly as a result of epigenetic regulation in the StAR promoter region.

Toxic effects of cypermethrin on the male reproductive system: with emphasis on the androgen receptor

The 15-day intact adult male assay was used to investigate the reproductive toxicity of cypermethrin. We also evaluated the contributions of the androgen receptor (AR) to cypermethrin-induced reproductive impairments. Sixty adult male Sprague-Dawley rats were randomly divided into five groups and treated with different doses of cypermethrin (0, 6.25, 12.5, 25 and 50 mg kg(-1) per day) by oral gavage for 15 days. After the rats were sacrificed, the testes, epididymides, seminal vesicles and prostates were excised and weighed. One testis was frozen to be used for daily sperm production. Another testis was processed for AR immunohistochemical analysis and electron microscopic observation. We found that the weights of prostates were significantly decreased in cypermethrin treatment at doses of 25 and 50 mg kg(-1) per day. Rats treated with cypermethrin at 50 mg kg(-1) per day exhibited a significant reduction in testicular daily sperm production. Seminiferous tubule changes were noted, including atrophy and distorted seminiferous tubules, reduction and deformation of spermatogonia, spermatocyte and disordered arrangement of spermatoblasts. Ultrastructural changes were found in cypermethrin-treated groups with disrupted cellular junctions, abnormal morphology of the nucleus, necrosis of spermatogonia spermatocytes and Sertoli cells. To clarify the possible mechanism, AR expression and the serum levels of testosterone were assayed. AR levels were significantly reduced in the rats treated with cypermethrin and the serum levels of testosterone were reduced in cypermethrin treatment at a dose of 50 mg kg(-1) per day. These data suggested that cypermethrin can induce impairments of the structure of seminiferous tubules and spermatogenesis in the male rats. The impairments can be attributed to the reduced AR expression.

Adverse effects of neonatal exposure to 3,3′,4,4′,5,5′‐hexachlorobiphenyl on hormone levels and testicular function in male Sprague–Dawley rats

In this study, we investigated the time-course changes of hormone levels and sperm numbers in male Sprague-Dawley (SD) rats after neonatal exposure to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169). Neonatal rats were given (through oral gavages) doses of 0, 0.025, 0.25, or 0.5 mg/kg-day of PCB169 in corn oil from postnatal day 1 (PND1) to PND7. The rats were sacrificed at PND8, PND21, and PND90. PCB169 exposure was confirmed by the marked induction of liver CYP1A1 mRNA expression at these three time points. The testicular daily sperm production and the sperm counts of the epididymis cauda significantly decreased at PND90 compared to that of control. Although reductions in serum thyroxine and triiodothyronine levels occurred at all these three time points and at both PND21 and PND90, respectively, the mRNA expression of testicular thyroid hormone receptor α1 was suppressed significantly only at PND8. The serum and testicular testosterone (T) levels declined markedly at PND90 compared to the controls, but there was no effect at PND21. The mRNA expression of testicular steroidogenic factor 1 was inhibited markedly at the three time points, whereas those of StAR, P450c17, P450scc, and 3β-HSD were suppressed significantly only at PND90 relative to the controls. PCB169 treatment had no effects on pituitary follicle-stimulating hormone and luteinizing hormone levels and on their receptors' expression in the testes. These results indicate that neonatal exposure to PCB169 damages hormone levels and testicular function in the long-term, resulting in persistent hypothyroidism and decreases in adult T levels and sperm counts.

Toxic effect of neonatal exposure to 2,2',4,4',5,5'-hexa-chlorobiphenyl on spermatogenesis in rats

To determine the long-term testicular effect after neonatal exposure to 2,2', 4,4',5,5'-hexa-chlorobiphenyl (PCB153). On birth day (Postnatal day 0, PNDO), the Sprague-Dawley (SD) male rats were mixed together and divided into 12 pups/litter. At PND1, the rats were grouped randomly into control and treatment groups according to different litters, 24 pups/group. They were treated by oral gavage with PCB153 in corn oil at doses of 0, 0.025, 0.250 and 2.500 mg/kg BW-day from PNDI to PND7. The rats were sacrificed at PND8 and PND90 by anesthesia. The testes were collected and weighed for histological examination and daily sperm production at PND8 or/and PND90. The epididymidis and the epididymidis cauda also were collected and weighed for determination the sperm counts at PND90. The body weight of 2.500 mg/kg dose group was decreased significantly from PND3 to PND8 compared with that of control (P < 0.05). At PND8, the loose structure in seminiferous cord and the spermatogonia with enlarged volume and detached from the cord were observed in 2.500 mg/kg dose group by light microscope and electronic microscopy. With the increase of exposure doses, the testicular daily sperm production (DSP) and the sperm counts of epididymidis cauda were decreased in dose-dependent manner at PND90. The DSP in 0.250 mg/kg [30 x 10(6)/testis(g)] and 2.500 mg/kg [18 x l0(6)/testis(g)] dose groups were significantly reduced compared with that of control [36 x 10(6)/testis(g)] (P < 0.05). And there was a significant reduction in the sperm counts of epididymidis cauda in 0.250 mg/kg [42 x 10(7)/epididymidis cauda (g)] and 2.500 mg/kg [18 x 10(7)/epididymidis cauda (g)] dose groups compared with that of control [51 x 10(7)/epididymidis cauda (g)] (P < 0.05). The spermatogenesis of adult testis is disturbed, which causes the decrease in the testicular DSP and the sperm counts of epididymidis cauda after neonatal exposure to PCB153. The long-term damage in male reproductive function is caused by neonatal exposure to chemicals.

119. Over-expression of activin βC in vivo reveals a role in male fertility

Introduction: Activin βC subunit antagonises the formation and bioactivity of activin A via intracellular heterodimerisation and decreases activation of the activin signalling pathway (Mellor et al. 2003). Therefore the activin βC subunit heterodimers provide a new mechanism of regulating activin levels. Vedja and colleagues over-expressed the activin βC subunit in malignant liver cell lines, which subsequently displayed inhibition of cell proliferation and induction of apoptosis (Vedja et al. 2003). Conversely, Wada et al. demonstrated that treatment with hr-activin C stimulates growth of a liver cell line (Wada et al. 2004). These recent (and contradictory) reports about the in vitro activity of activin βC have prompted us to examine the in vivo role of activin βC by creating a transgenic mouse over-expressing the βC activin subunit. Methods: The full-length human cDNA under the control of a CMV promoter was incorporated into the genome of three founder C57/B6 mice. Genotyping was performed by both Southern and PCR. Mice were monitored weekly and culled at 14–16 weeks (adult). Blood was collected by cardiac puncture, organs were weighed and a portion fixed in Bouin’s or frozen for subsequent RNA and protein extraction. Daily sperm production (DSP) was determined by standard methods. Sertoli and germ cell number will be determined using the optical disector (sic) stereological technique in Bouin’s fixed resin sections. Proliferation and apoptosis will be examined using PCNA and TUNEL respectively. Activin A was assessed by ELISA, while FSH, LH, follistatin and total inhibin were determined by RIA. Results and conclusions: Over-expression of activin-βC resulted in decreased circulating activin A (P &lt; 0.005 TG1, P &lt; 0.05 TG2 and P = 0.08 TG3), a progressive age-related decrease in litter sizes (9.3 WT v. 6.3 TG1, 5.8 TG2 and 4.5 TG3; P &lt; 0.005 v. WT) and testicular DSP (P &lt; 0.05). These data support the hypothesis that βC is a novel in vivo regulator and is the first indication of a role for activin-βC in male fertility. This novel mouse model will significantly advance our understanding of the in vivo role of activin-βC. (1)Mellor et al. (2003). Endocrinology 144, 4410–4419.(2)Vejda et al. (2003). Carcinogenesis 24, 1801–1809.(3)Wada et al. (2004). Am. J. Physiol. Endocrinol. Metab. 287, E247–E254.

Testicular toxicity of dietarily or parenterally administered bisphenol A in rats and mice

Male Crj:Wistar rats, HsdHot:Holtzman SD rats, Crj:CD-1(ICR) mice and C57BL/6CrSlc mice were administered bisphenol A (BPA) in the diet at a level of 0 (control) and 0.25% for 8 weeks. Daily BPA intake was about 200 and 400 mg/kg for rats and mice, respectively. No conspicuous signs of general or reproductive toxicity were observed after administration in any strain of these animals. Serum testosterone concentrations were not decreased in BPA-fed rats and mice. Successive subcutaneous administration of BPA at a dose of 200 mg/kg/day for 4 weeks significantly decreased the testis, epididymis, prostate and seminal vesicle weights, and the testicular daily sperm production in Jcl:Wistar rats. Successive intraperitoneal administration of BPA at a dose of 20 mg/kg/day for 4 weeks decreased the prostate and seminal vesicle weights but not the testis or epididymis weights. An intraperitoneal dose of 2 mg BPA/kg/day did not cause any toxicity. These results indicate that dietarily administered BPA is less toxic to most strains of rats and mice, and the maximum non-toxic dose and/or minimum toxic dose may be about 200 mg/kg/day. Subcutaneous or intraperitoneal BPA is much more toxic on male reproductive and sex accessory organs than dietary.

Reproductive Capacity of Dairy Bulls. VIII. Direct and Indirect Measurement of Testicular Sperm Production1,2,3

Summary It was desired to determine testicular daily sperm production (DSP) and compare it with daily sperm output (DSO). Two independent calculations of DSP for each testis from eight 2-yr-old and four mature Holstein bulls were based on counts of gonadal sperm reserves and on quantitative testicular histology. Limitations of the proposed methods and formulas were discussed. The duration of spermatogenesis in bulls was calculated to be 60 days. The average DSP values calculated from gonadal sperm reserves and histological data were 9.98 and 12.76 billion per bull. The differences between DSP values for the 24 testes by the two methods of calculation were highly significant, but the two values were correlated (+0.81). However, testis weight accounted for most of the relationship, since the correlation was not significant when calculated on the basis of DSP per gram of testis (+0.28). Since the validity and repeatability of these methods has not been established, the two DSP values were averaged and ranged from 7.7 to 13.2 billion for eight bulls of similar age and body weight. The DSO during collection of six ejaculates weekly with intensive sexual preparation (6×) also was obtained for the 12 bulls. About five months elapsed between collection of DSO data and postslaughter determination of DSP. The DSO on 6× averaged 4.81 ± 0.35 billion, or only 42% of the mean DSP of 11.49 ± 0.64 billion. However, the highly significant correlation between DSP and DSO on 6× (+0.87) suggests that DSP can be estimated in individual living bulls by collecting semen at a suitable high ejaculation frequency.