Pfaffia glomerata polyploid accession compromises male fertility and fetal development.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata (Spreng.) Pedersen has traditionally been used as a tonic and a stimulant by the Brazilian population. It shows higher biomass accumulation and production of secondary compounds, such as the phytosterol 20-hydroxyecdysone. AIMS: The present study aimed to evaluate the effects of the hydroalcoholic extract of the root of tetraploid P. glomerata (BGEt) on testicular parenchyma, and its implications on fertility. MATERIAL AND METHODS: Adult Swiss mice were divided as: control (water) and sildenafil citrate (7 mg/kg), BGEt at 100, 200, and 400 mg/kg, and BGEtD 200 mg/kg (treated with BGE every three days). Males (n = 4/group) were mated with normal untreated adult females to assess fertility rates, while other animals (n = 6/group) were euthanized for testis, epididymis, and oxidative stress analyses. RESULTS: Increase in tubule diameter and epithelium height in the discontinuous group, in addition to an increase in the proportion of tubules with moderate pathologies was observed. The pre-implantation loss was lower in all treated groups. The post-implantation loss was significantly increased in all treated groups, except for the lowest BGEt dose. BGEt intake caused a decrease in daily sperm production, along with the number and quality of sperm in the epididymis. Changes were observed in protein carbonylation and hydrogen peroxide and nitric oxide levels, characterizing oxidative stress. CONCLUSIONS: The hydroalcoholic extract of P. glomerata tetraploid altered sperm and testicular parameters, compromising embryonic development after implantation.

Alterations in the testicular parenchyma of Foxn1+/- and Foxn1-/- adult mice.

Nude mice carry an autosomal recessive mutation in the Foxn1 gene and therefore are homozygous recessive animals (Foxn1 -/-). The fertility rate of homozygous male (Foxn1-/- ) is low, which seems to be related to the delay in the production of gametes at the beginning of sexual maturity. The present study evaluated the structural and organizational aspects of the testicles of homozygous and heterozygous offspring related to the Foxn1 gene in mice, describing its implications on spermatogenesis. Adult males Balb/c, Foxn1+/- and Foxn1-/- mice were used. Testes and epididymis were harvested for histological, biochemical, and sperm transit analyses. Gonadal weight was significantly lower in Foxn1+/- and Foxn1-/- animals, the same behavior was noticed for the activity of antioxidant enzymes. In addition, tubular parameters such as epithelial proportion, length, and area, as well as germ and Leydig cell's populations were significantly reduced in the aforementioned groups, leading to lower sperm production. In conclusion, our results indicate the importance of the Foxn1 in Leydig cell's function, reflecting in the preservation of spermatogenesis, thus in germ cell's population and sperm cell production.

Deleterious effects of Pfaffia glomerata (Spreng.) Pedersen hydroalcoholic extract on the seminiferous epithelium of adult Balb/c mice.

Several plant species such as Pfaffia glomerata are widely used in traditional Brazilian medicine as stimulants and aphrodisiacs. In this regard, the aim of our study was to explore the effects of the long-term intake of the hydro-alcoholic root extract of P glomerata on the germ and somatic cells within the seminiferous tubules in adult Balb/c mice. The experimental groups were placed as: controls (water and DMSO), and treated with 300 and 400 mg/kg of the root extract. The number of germ and somatic cells, the proportion of pathological seminiferous tubules, and the germ cell apoptotic levels were evaluated. The volume and proportion of the seminiferous epithelium was decreased after the extract intake due to the increased germ cell apoptotic levels. Vacuolization of Sertoli cell cytoplasm was observed widely in pathological tubules, along with fully disorganized epithelia, showing multinucleated cells, which lead to decreased daily sperm production. Taken together, our results indicate that long-term intake of the P glomerata caused deleterious effects on spermatogenesis by inducing apoptosis and altering the seminiferous tubule's epithelial dynamics.

Subacute Testicular Toxicity to Cadmium Exposure Intraperitoneally and Orally.

The toxic effects of cadmium (Cd) on reproductive parameters are widely described in the literature. Experimental models often make use of the intraperitoneal route (i.p.), although human intoxication occurs preferentially by the oral route and can be continuous. However, little is known about the effect of Cd administration routes on the testicular structure. Thus, this study investigated the testicular impact of Cd exposure comparing both i.p. and oral routes, both single dose (SD), in addition to the oral route in fractional doses (FD). Swiss adult male mice received CdCl2 1.5 mg/kg i.p., 30 mg/kg oral SD, and 4.28 mg/kg oral FD for 7 consecutive days. The Cd bioaccumulation was observed in all routes, mainly in the oral FD route. The concentrations of testicular Ca and Cu decreased in all animals exposed to Cd, while Zn and Mn decreased only in the i.p. route. Testicular SOD activity was reduced in both routes of oral administration, while CAT increased in the i.p. route, and GST increased in all animals exposed to Cd. Changes in the tubular parameters and cell viability were observed in both routes of Cd administration but were more intense in the oral route, mainly in the FD. Serum testosterone concentration was reduced in both routes of oral administration. Tubular damage, such as the vacuolization of the seminiferous epithelium, germ cell detachment, and seminiferous tubule degeneration, occurred in all groups exposed to Cd. Therefore, the oral Cd administration presented greater potential to promote testicular damage, mainly when the metal was given in a fractionated way.

Spermatogenic dynamics of the spiny rat Kannabateomys amblyonyx (Wagner, 1845) (Rodentia, Echimyidae).

Rodents are distributed worldwide, playing important ecological roles regarding preservation of forest areas. Thus, the study of their reproductive biology is a key for conservation initiatives that prevent extinction and/or improve species management. The present study aimed to describe the spermatogenic dynamics of the spiny rat Kannabateomys amblyonyx, an endemic species from Atlantic Rainforest areas, in Brazil. The average body weight was 418.43g with a gonadosomatic index of 0.41%. The testicular parenchyma organization followed the pattern described for other rodent species, with a large amount of seminiferous tubules occupying 93.57% of the organ, in a total of 26.04m per testicle. Stage I of the seminiferous tubule cycle was the most frequent in K. amblyonyx, while stage IV the most scarce. Each tubular section in stage I showed 0.47 type-A spermatogonia, 11.78 primary spermatocytes in pre-leptotene, 3.81 in zygotene and 14.31 in pachytene, whereas 32.19 cells were round spermatids and 6.23 were Sertoli cells. From these results it was possible to determine the sperm reserve of 274.49×106 cells per gram of testis. The mitotic and meiotic indexes were 25.06 and 2.25 cells, respectively, whereas the spermatogenic yield was 69.73 cells. Those findings are significant since this is the first study regarding the reproductive aspects of the only Echimyidae species in Brazil, which shows a monogamous mating system.

Transient Hypothyroidism: Dual Effect on Adult-Type Leydig Cell and Sertoli Cell Development.

Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations.

Influence of three different histological methods on the morphology and morphometrical data in human testis.

Coagulant fixatives and paraffin embedding were widely used in the past for histomorphometrical evaluations of the human testis under physiological and pathological conditions. However, new methods are applied nowadays using better combinations of fixatives and plastic resins as embedding media, improving cell and tissue structural preservation. In an attempt to compare old and new data, the present study evaluated histomorphometrical data obtained from human testis after three different histological processing methods: Bouin/paraplast, glutaraldehyde/glycol methacrylate and glutaraldehyde/araldite. The morphometrical parameters were not affected by glutaraldehyde fixation after both resin embedding (methacrylate or araldite). On the other hand, Bouin/paraplast embedding lead to tissue shrinkage, which could give rise to misinterpretations on the measurements performed. Since some germ and somatic cells recognition do not depend upon high resolution techniques, counting of such cell types could be performed even using routine Bouin/paraplast protocols. Thus, the morphometrical analyses relying on cell recognition were not affected by the methods here applied, however, when metric measurements were applied, the obtained results could not be promptly compared. However, if the study requires confident spermatogonial identification for kinetics evaluation, glutaraldehyde/araldite processing is highly recommended.

Evaluation of the cell population of the seminiferous epithelium and spermatic indexes of the bat Sturnira lilium (Chiroptera: Phyllostomidae).

Due to the scarcity of information about patterns of spermatogenesis in bats, this study aimed to provide information on the testicular activity of the bat Sturnira lilium along the annual seasons. Thus, a series of morphometrical and stereological analyses were made using the testes of adult S. lilium in order to achieve a better understanding of the sperm production dynamics. Light and transmission electron microscopy analyses were performed in testicular fragments of animals captured during dry and rainy seasons. The testes followed the pattern of organization described for other mammals, and there were no morphological differences between organs collected either in dry or in rainy seasons. Each tubular cross-section in stage 1 was made of 0.5 type-A spermatogonia, 4.4 primary spermatocytes in preleptotene/leptotene, 3.7 in zygotene, 11.9 in pachytene, 35.6 round spermatids and 8.5 Sertoli cells. The mitotic and meiotic indexes were 15.4 and 2.9 cells, respectively, while the spermatogenesis yield was 68.7 cells. The testicular sperm reserves was 37.61×10(6) cells, and daily sperm production per gram of testis averaged 209.68×10(6) cells, both highest averages occurring in the rainy season. S. lilium male bats have a continuous reproductive pattern, high spermatogenesis yield and low support capacity by the Sertoli cells.

Heteropterys tomentosa (A. Juss.) infusion counteracts Cyclosporin a side effects on the ventral prostate.

BACKGROUND: Cyclosporin A (CsA) is an immunosuppressive drug widely used in treatment of auto-immune diseases or after organ transplants. However, several side effects are commonly associated with CsA long term intake, some regarding to loss of reproductive organ function due to oxidative damage. Considering that phytotherapy is an important tool often used against oxidative stress, we would like to describe the beneficial effects of Heteropterys tomentosa intake to minimize the damage caused by CsA to the ventral prostate tissue of Wistar rats under laboratorial conditions. METHODS: Thirty adult Wistar rats (Rattus norvegicus albinus) were divided into: control group (water); CsA group (Cyclosporin A); Ht group (H. tomentosa infusion) and CsA + Ht group (CsA and H. tomentosa infusion). Plasmic levels of hepatotoxicity markers, triglycerides, cholesterol and glucose were quantified. The ventral prostate tissue was analyzed under light microscopy, using stereological, morphometrical and immunohistochemical techniques. RESULTS: H. tomentosa did not cause any alterations either of the plasmic parameters or of the ventral prostate structure. CsA caused alterations of GOT, total and indirect bilirubin, cholesterol, triglycerides and glucose levels in the plasma; CsA-treated rats showed alterations of the ventral prostate tissue. There were no alterations regarding the plasma levels of GOT, triglycerides and glucose of CsA + Ht animals. The same group also showed normalization of most of the parameters analyzed on the ventral prostate tissue when compared to the CsA group. The treatments did not alter the pattern of AR expression or the apoptotic index of the ventral prostate epithelium. CONCLUSIONS: The results suggest a protective action of the H. tomentosa infusion against the side effects of CsA on the ventral prostate tissue, which could also be observed with plasmic biochemical parameters.

More resistant tendons obtained from the association of Heteropterys aphrodisiaca and endurance training.

BACKGROUND: Popular Brazilian medicine uses Heteropterys aphrodisiaca infusion as a tonic or stimulant, for the treatment of nervous debility and breakdown and for muscle and bone weakness. This study investigated the effects of Heteropterys aphrodisiaca infusion on the tendon properties and extracellular matrix of rats under endurance training. METHODS: Wistar rats were grouped as follows: CS- control sedentary, HS- H. aphrodisiaca sedentary, CT-control trained, HT- H. aphrodisiaca trained. The training protocol consisted in running on a motorized treadmill, five times a week, with weekly increase in treadmill speed and duration. Control groups received water while the HS and HT groups received H. aphrodisiaca infusion, daily, by gavage for the 8 weeks of training. Achilles tendons were frozen for biochemical and biomechanical analysis or preserved in Karnovsky's fixative, then processed for histomorphological analysis with light microscopy. RESULTS: Biomechanical analysis showed significant increase in maximum load, maximum stress, modulus of elasticity and stiffness of the HT animals' tendons. The metalloproteinase-2 activity was reduced in the HT group. The compression region of HT animals' tendons had a stronger and more intense metachromasy, which suggests an increase in glycosaminoglycan concentration in this region of the tendon. The most intense birefringence was observed in both compression and tension regions of HT animals' tendons, which may indicate a higher organizational level of collagen bundles. The hydroxyproline content increased in the HT group. CONCLUSIONS: The association of endurance training with H. aphrodisiaca resulted in more organized collagen bundles and more resistant tendons to support higher loads from intense muscle contraction. Despite the clear anabolic effects of Heteropterys aphrodisiaca and the endurance exercise association, no side effects were observed, such as those found for synthetic anabolic androgenic steroids.

Association of the infusion of Heteropterys aphrodisiaca and endurance training brings spermatogenetic advantages.

The species Heteropterys aphrodisiaca is commonly used as a stimulant by popular medicine in the Cerrado, a savanna-like biome, Brazil. Recent studies have proved its protective effects on testes of animals submitted to treatment using Cyclosporine A, as well as its stimulus effect in increasing testosterone secretion. Therefore, the present study was designed to analyze whether the association of the plant infusion and endurance exercise could potentiate the stimulating effect. The animals were separated into 4 groups: two control (sedentary and trained) receiving water and two treated (sedentary and trained) receiving the plant infusion daily (104 mg/day). The proportion of the seminiferous tubule compartment and interstitium was analyzed. Within the seminiferous epithelium, the number of Sertoli and germ cells were counted in order to evaluate whether the treatment would alter the spermatogenic dynamics, analyzing: the spermatogenic yield, the mitotic and meiotic indexes, the total number of germ cells and the Sertoli cell support capacity. Trained and treated animals showed increased spermatogenic yield and spermatogonia mitosis, and no significant differences in apoptotic indexes. Despite the results showing the same pattern regarding yield and mitotic index, the meiotic index was higher in the sedentary/treated group. Therefore, the H. aphrodisiaca infusion increased both the testosterone production and the spermatogonia mitosis, thus increasing the spermatogenic yield.

Association of the infusion of Heteropterys aphrodisiaca and endurance training brings spermatogenetic advantages

The species Heteropterys aphrodisiaca is commonly used as a stimulant by popular medicine in the Cerrado, a savanna-like biome, Brazil. Recent studies have proved its protective effects on testes of animals submitted to treatment using Cyclosporine A, as well as its stimulus effect in increasing testosterone secretion. Therefore, the present study was designed to analyze whether the association of the plant infusion and endurance exercise could potentiate the stimulating effect. The animals were separated into 4 groups: two control (sedentary and trained) receiving water and two treated (sedentary and trained) receiving the plant infusion daily (104mg/day). The proportion of the seminiferous tubule compartment and interstitium was analyzed. Within the seminiferous epithelium, the number of Sertoli and germ cells were counted in order to evaluate whether the treatment would alter the spermatogenic dynamics, analyzing: the spermatogenic yield, the mitotic and meiotic indexes, the total number of germ cells and the Sertoli cell support capacity. Trained and treated animals showed increased spermatogenic yield and spermatogonia mitosis, and no significant differences in apoptotic indexes. Despite the results showing the same pattern regarding yield and mitotic index, the meiotic index was higher in the sedentary/treated group. Therefore, the H. aphrodisiaca infusion increased both the testosterone production and the spermatogonia mitosis, thus increasing the spermatogenic yield.

The effects of Tynnanthus fasciculatus (Bignoniaceae) infusion on testicular parenchyma of adult Wistar rats.

Traditional medicine provides strong guidance for scientific experiments involving plant products used by the Brazilian people. The species "cipó-cravo" (Tynnanthus fasciculatus) is a plant commonly used either to combat indigestion and stomach aches, or as a general stimulant and aphrodisiac. In this study, the effects of "cipó-cravo" infusion were investigated within the testicular parenchyma of adult Wistar rats. Rats were divided into 3 groups: a control (distilled water) and two treated groups, which received the plant infusion (100 and 200mg/animal/day). The 200mg dose promoted a significant increase of the testicular parenchyma weight and of the volume and total length of the seminiferous tubules, as well as in total daily sperm production and sperm production per gram of testis.

The effects of Tynnanthus fasciculatus (Bignoniaceae) infusion on testicular parenchyma of adult Wistar rats

Traditional medicine provides strong guidance for scientific experiments involving plant products used by the Brazilian people. The species "cipó-cravo" (Tynnanthus fasciculatus) is a plant commonly used either to combat indigestion and stomachaches, or as a general stimulant and aphrodisiac. In this study, the effects of "cipó-cravo" infusion were investigated within the testicular parenchyma of adult Wistar rats. Rats were divided into 3 groups: a control (distilled water) and two treated groups, which received the plant infusion (100 and 200mg/animal/day). The 200mg dose promoted a significant increase of the testicular parenchyma weight and of the volume and total length of the seminiferous tubules, as well as in total daily sperm production and sperm production per gram of testis.