Effect of Clomiphene Citrate on the Ultrastructure of Testis in Albino Rats

SUMMARY: The aim of the present work was to study the closer effect of clomiphene citrate on the ultrastructure of the testis of adult albino rats to provide a basis for optimizing this drug in the treatment of male infertility. The testes were removed from both groups under anesthesia and then prepared for examination by light using hematoxylin and eosin stains and a transmission electron microscope. Semithin sections were cut into 1 µm thick sections, stained with toluidine blue, and examined by light microscopy for a survey. The desired areas were placed in the center, and other areas were trimmed. Primary spermatocytes showed marked nuclear changes (pyknosis), and their nuclear membranes were ill-defined and disrupted. The cytoplasm showed widespread degeneration of mitochondria and lysosomes and focal degeneration of the rough endoplasmic reticulum compared with the control group. The spermatids were pale, and the two phases of spermatogenesis were distinctly identifiable in the control group but were confused in the treated group. Some spermatids had interrupted nuclear membranes, also containing degenerated mitochondria, focal fragmentation of rough endoplasmic reticulum, and free ribosomes. Spermatozoa in the treated group appeared deformed compared to the control, where they had deformed head caps. Leydig cells of the treated group have an irregularly shaped nucleus, with focal chromatin aggregation and peripheral chromatin condensation on the inner surface of the nuclear membrane. The observations of the present work indicate a possible causal relationship between testicular affection and ingestion of clomiphene citrate, which can be avoided by close medical observations using ultrasonography, semen analysis, or testicular biopsy to detect early malignant changes. Furthermore, the drug should not be used for more than three to six cycles and should be stopped for at least three cycles before reuse. When clomiphene citrate is ineffective in the treatment of male infertility, human menopausal gonadotropin (hMG) administration is typically selected. However, high-dose hMG therapy is associated with a variety of adverse effects. In this work, we report the success of a modified clomiphene citrate regimen in increasing sperm count without any hazards to the testicular tissue.

El objetivo del trabajo fue estudiar el efecto del citrato de clomifeno sobre la estructura de los testículos de la rata albina adulta, con la finalidad de determinar la mejor manera de utilizar este fármaco en el tratamiento de la infertilidad masculina. Los testículos se extrajeron bajo anestesia y para su análisis a través de microscopio de luz se tiñeron con HE. Además, las muestras fueron preparadas para su examen con microscopía electrónica de transmisión. Por otra parte, se cortaron secciones semifinas de 1 µm de espesor, se tiñeron con azul de toluidina y se examinaron mediante microscopía óptica. Los espermatocitos primarios mostraron cambios nucleares marcados (picnosis) y sus membranas nucleares estaban mal definidas y alteradas. En el grupo experimental las células presentaban el citoplasma con degeneración generalizada de las mitocondrias y de los lisosomas y una degeneración focal del retículo endoplásmico rugoso en comparación con el grupo control. Las espermátidas estaban pálidas y las dos fases de la espermatogénesis eran claramente identificables en el grupo control, pero se confundían en el grupo tratado. Algunas espermátidas tenían membranas nucleares interrumpidas, y también contenían mitocondrias degeneradas, fragmentación focal del retículo endoplásmico rugoso y ribosomas libres. Los espermatozoides del grupo tratado se presentaban deformados en comparación con el control. Las células de Leydig del grupo tratado presentaban un núcleo de forma irregular, con agregación focal de cromatina y condensación de cromatina periférica en la superficie interna de la membrana nuclear. Las observaciones del presente trabajo indican una posible relación causal entre la afección testicular y la ingestión de citrato de clomifeno, que puede evitarse mediante observaciones médicas minuciosas a través de ecografía, análisis de semen o biopsia testicular para detectar cambios malignos tempranos. Además, el medicamento no debiera ser usado durante más de tres a seis ciclos y debe suspenderse durante al menos tres ciclos antes de volver a usarlo. Cuando el citrato de clomifeno es ineficaz en el tratamiento de la infertilidad masculina, normalmente se selecciona la administración de gonadotropina menopáusica humana (hMG). Sin embargo, la terapia con hMG en dosis altas se asocia con una variedad de efectos adversos. En este trabajo, informamos el éxito de un régimen modificado con citrato de clomifeno para aumentar el recuento de espermatozoides sin riesgo para el tejido testicular.

Doxycycline hyclate may damage testicular germinal epithelium in Wistar rats

Broad-spectrum antimicrobial doxycycline acts as an inhibitor of protein synthesis and it is widely used in the clinical treatment of various infections by microorganisms that are sensitive to the drug, as well as in animal feed. Its liposolubility guarantees its high tissue bioavailability, being associated with several biochemical changes in the organism and potentially adverse effects on reproduction. This study aims to evaluate the effects of the action of doxycycline on spermatogenesis to provide a complete analysis of the tubular and interstitial compartments and to identify possible changes in the testicular parenchyma. Adult male Wistar rats were divided into three groups: one control (water), and two treated with doxycycline at the doses of 10mg/kg and 30mg/kg, for 30 days. After euthanasia and sample processing, the following parameters were evaluated: a) tubular diameter and height of the seminiferous epithelium; b) volumetric proportions (%) and volumes (mL) of the components of the testicular parenchyma; c) counting testicular germ cell populations; d) evaluation of cell viability. The results of the comparative evaluation between the experimental groups demonstrated a significant increase in the diameter and area of the tubular lumen and a reduction in the count of spermatogonia in the experimental group that received doxycycline hyclate at a dose of 30mg/kg. In the same experimental group, an increase in the overall yield of spermatogenesis was found as a consequence of the increase in the mitotic index.

Short-term exposure to gossypol causes reversible reproductive toxicity and nephrotoxicity in mice / 南方医科大学学报

OBJECTIVE@#To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible.@*METHODS@#Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity.@*RESULTS@#Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity.@*CONCLUSIONS@#Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.

Ubiquitin protein E3 ligase ASB9 suppresses proliferation and promotes apoptosis in human spermatogonial stem cell line by inducing HIF1AN degradation

BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.

Gonadal characterization of the Amazonian fish Serrapinnus kriegi (Characidae: Cheirodontinae) / Caracterização gonadal do peixe Amazônico Serrapinnus kriegi (Characidae: Cheirodontinae)

The knowledge of the testicular and ovarian morphology of a particular fish species is of paramount importance. Such analyze enables the development of studies and techniques aiming the improvement of their reproduction, management, commercialization and even their conservation. This study performed the ovarian and testicular characterization of the ornamental Amazon fish Serrapinnus kriegi. A total of three males and three females had their gonads analyzed by optical microscopy. Females present ovaries filled with oocytes in asynchronous development, indicating partial spawning in the species. Moreover, the micropyle and micropilar cell formation was observed in primary growing oocytes, representing a precocious oocyte development; and the zona radiata in the final vitellogenic oocytes is thicker than other related species, evidencing the development of a better protection to the embryos in function of the waters’ turbulence that characterize it spawning sites in the Amazonian streams. The male specimens’ present anastomosed tubular testes with unrestricted spermatogonia spread along the entire seminiferous tubules. The present data elucidate the dynamic of spermatogenesis and oogenesis of an ornamental Amazonian species, through the description of the male and female germ cells development.

O conhecimento da morfologia testicular e ovariana de uma determinada espécie de peixe é de suma importância, pois através destas análises é possível o desenvolvimento de estudos e técnicas visando o melhoramento de sua reprodução, manejo e comercialização e até mesmo auxiliar em sua conservação. Este estudo realizou a caracterização ovariana e testicular do peixe Amazônico ornamental Serrapinnus kriegi. Um total de três machos e três fêmeas tiveram suas gônadas analisadas através de microscopia óptica. As fêmeas apresentam ovários preenchidos por oócitos em desenvolvimento assincrônico, indicando desova parcelada da espécie. Além disso, observou-se a formação de micrópila e célula micropilar em oócitos em crescimento primário, representando o desenvolvimento precoce do oócito; a zona radiata nos oócitos vitelogênicos finais é mais espessa em comparação a outras espécies relacionadas, evidenciando o desenvolvimento de uma melhor proteção aos embriões, em função das águas turbulentas que caracterizam seu local de desova nos córregos amazônicos. Os machos apresentam testículos do tipo tubular anastomosado com espermatogônias irrestritas, espalhadas por todo o túbulo seminífero. Os dados apresentados elucidam a dinâmica da espermatogênese e oogênese de uma espécie de peixe ornamental amazônica, por meio da descrição das células germinativas masculinas e femininas.

Loss-of-function CFTR p.G970D missense mutation might cause congenital bilateral absence of the vas deferens and be associated with impaired spermatogenesis / 亚洲男科学杂志(英文版)

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.

Characterization of the protein expression and localization of hnRNP family members during murine spermatogenesis / 亚洲男科学杂志(英文版)

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Immature rat testis sustained long-term development using an integrative model

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

Effects of melatonin on both testicular regeneration and recovery of spermatogenesis in busulfan-treated rats

Abstract Testicular damage is one of the most hazardous effects of chemotherapy as it is frequently associated with oligozoospermia and azoospermia. This study aimed at evaluating the protective effect of melatonin in a rat model of busulfan-induced testicular injury. Rats were divided into four groups: control, melatonin, busulfan, busulfan plus melatonin. After 15 days, the semen was collected from the epididymis and testes were assessed. Sperm removed from cauda epididymis and analyzed for sperm count and viability. Testis tissues were also removed, fixed in formalin and were embedded in paraffin. Sections of testis tissue were stained with hematoxylin-eosin for histological examination and prepared for TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay to detect apoptosis and PCNA (proliferating cell nuclear antigenassay) to detect proliferation cells. Serum and testes supernatants were separated to detect testosteron level and oxidative stress parameters. In histological examination, degenerative changes in seminiferous tubules were observed in the experimental groups. In biochemical examination, the total oxidant status (TOS) levels in Busulfan group were significantly higher than in the control group while the total antioxidant status (TAS) levels of all the groups were similar. In conclusion, the beneficial properties of melatonin treatment by its potent anti-oxidants may reduce adverse effects of chemotherapy in the reproductive system in a rodent system.

Genetic pathogenesis of acephalic spermatozoa syndrome: past, present, and future / 亚洲男科学杂志(英文版)

Acephalic spermatozoa syndrome (ASS) is one of the most severe spermatogenic failures of all infertility in men. The cognition of ASS has experienced a tortuous process. Over the past years, with the in-depth understanding of spermatogenesis and the emergence of new genetic research technologies, the unraveling of the genetic causes of spermatogenic failure has become highly active. From these advances, we established a genetic background and made significant progress in the discovery of the genetic causes of ASS. It is important to identify pathogenic genes and mutations in ASS to determine the biological reasons for the occurrence of the disease as well as provide genetic diagnosis and treatment strategies for patients with this syndrome. In this review, we enumerate various technological developments, which have made a positive contribution to the discovery of candidate genes for ASS from the past to the present. Simultaneously, we summarize the known genetic etiology of this phenotype and the clinical outcomes of treatments in the present. Furthermore, we propose perspectives for further study and application of genetic diagnosis and assisted reproductive treatment in the future.

Comparison of intracytoplasmic sperm injection (ICSI) outcomes in infertile men with spermatogenic impairment of differing severity / 亚洲男科学杂志(英文版)

The extent of spermatogenic impairment on intracytoplasmic sperm injection (ICSI) outcomes and the risk of major birth defects have been little assessed. In this study, we evaluated the relationship between various spermatogenic conditions, sperm origin on ICSI outcomes, and major birth defects. A total of 934 infertile men attending the Center for Reproductive Medicine of Ren Ji Hospital (Shanghai, China) were classified into six groups: nonobstructive azoospermia (NOA; n = 84), extremely severe oligozoospermia (esOZ; n = 163), severe oligozoospermia (sOZ, n = 174), mild oligozoospermia (mOZ; n = 148), obstructive azoospermia (OAZ; n = 155), and normozoospermia (NZ; n = 210). Rates of fertilization, embryo cleavage, high-quality embryos, implantation, biochemical and clinical pregnancies, abortion, delivery, newborns, as well as major birth malformations, and other newborn outcomes were analyzed and compared among groups. The NOA group showed a statistically lower fertilization rate (68.2% vs esOZ 77.3%, sOZ 78.0%, mOZ 73.8%, OAZ 76.6%, and NZ 79.3%, all P < 0.05), but a significantly higher implantation rate (37.8%) than the groups esOZ (30.1%), sOZ (30.4%), mOZ (32.6%), and OAZ (31.0%) (all P < 0.05), which was similar to that of Group NZ (38.4%). However, there were no statistically significant differences in rates of embryo cleavage, high-quality embryos, biochemical and clinical pregnancies, abortions, deliveries, major birth malformations, and other newborn outcomes in the six groups. The results showed that NOA only negatively affects some embryological outcomes such as fertilization rate. There was no evidence of differences in other embryological and clinical outcomes with respect to sperm source or spermatogenic status. Spermatogenic failure and sperm origins do not impinge on the clinical outcomes in ICSI treatment.

Biallelic mutations in spermatogenesis and centriole-associated 1 like (SPATC1L) cause acephalic spermatozoa syndrome and male infertility / 亚洲男科学杂志(英文版)

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

Ameliorative effects of morel mushroom (Morchella esculenta) against Cadmium-induced reproductive toxicity in adult male rats / Efeitos benéficos do cogumelo morel (Morchella esculenta) contra a toxicidade reprodutiva induzida por cádmio em ratos machos adultos

Cadmium (Cd) is one of the major toxicants, which affects human health through occupational and environmental exposure. In the current study, we evaluated the protective effects of morel mushrooms against Cd-induced reproductive damages in rats. For this purpose, 30 male rats were divided into 6 groups (n=5/group), the first group served as the control group, second group was treated with an intraperitoneal (i.p) injection of 1 mg/kg/day of Cd. Third and fourth groups were co-treated with 1 mg/kg/day of Cd (i.p) and 10 and 20 mg/kg/day of morel mushroom extract (orally) respectively. The final 2 groups received oral gavage of 10 and 20 mg/kg/day of morel mushroom extract alone. After treatment for 17 days, the animals were euthanized, and testes and epididymis were dissected out. One testis and epididymis of each animal were processed for histology, while the other testis and epididymis were used for daily sperm production (DSP) and comet assay. Our results showed that Cd and morel mushrooms have no effect on animal weight, but Cd significantly decreases the DSP count and damages the heritable DNA which is reversed in co-treatment groups. Similarly, the histopathological results of testes and epididymis show that morel mushrooms control the damage to these tissues. Whereas the morel mushroom extract alone could enhance the production of testosterone. These results conclude that morel mushrooms not only control the damage done by Cd, but it could also be used as a protection mechanism for heritable DNA damage.

O cádmio (Cd) é um dos principais tóxicos, que afeta a saúde humana por meio da exposição ocupacional e ambiental. No presente estudo, avaliamos os efeitos protetores dos cogumelos morel contra os danos reprodutivos induzidos pelo Cd em ratos. Para tanto, 30 ratos machos foram divididos em 6 grupos (n = 5 / grupo); o primeiro grupo serviu de controle, o segundo grupo foi tratado com injeção intraperitoneal (i.p) de 1 mg / kg / dia de Cd. O terceiro e o quarto grupos foram cotratados com 1 mg / kg / dia de Cd (i.p) e 10 e 20 mg / kg / dia de extrato de cogumelo morel (por via oral), respectivamente. Os dois grupos finais receberam gavagem oral de 10 e 20 mg / kg / dia de extrato de cogumelo morel sozinho. Após o tratamento por 17 dias, os animais foram sacrificados e os testículos e o epidídimo foram dissecados. Um testículo e epidídimo de cada animal foram processados para histologia, enquanto o outro testículo e epidídimo foram usados para produção diária de esperma (DSP) e ensaio cometa. Nossos resultados mostraram que os cogumelos Cd e morel não têm efeito sobre o peso do animal, mas o Cd diminui significativamente a contagem de DSP e danifica o DNA hereditário, que é revertido em grupos de cotratamento. Da mesma forma, os resultados histopatológicos dos testículos e do epidídimo mostram que os cogumelos morel controlam os danos a esses tecidos. Considerando que o extrato de cogumelo morel sozinho pode aumentar a produção de testosterona. Esses resultados concluem que os cogumelos morel não apenas controlam os danos causados pelo Cd, mas também podem ser usados como um mecanismo de proteção para danos hereditários ao DNA.

Efecto del consumo de marihuana sobre los parámetros espermáticos humanos: Aproximación in vivo / Effect of Marijuana Use on Human Semen Parameters: In Vivo Approach

La evidencia sugiere que la exposición a sustancias psicoactivas se relaciona con alteraciones en la espermatogénesis que afectan la calidad espermática. El objetivo de este trabajo fue determinar los parámetros espermáticos en consumidores habituales de cigarrillos de marihuana. Se analizaron muestras seminales de 42 consumidores activos de cigarrillos de marihuana y de 16 voluntarios no consumidores de marihuana. Mediante un análisis de semen, se determinaron los parámetros seminales convencionales (viabilidad, movilidad, morfología, y concentración de los espermatozoides) siguiendo los lineamientos establecidos por la Organización Mundial de la Salud (OMS). Adicionalmente, se evaluó la capacidad antioxidante del plasma seminal mediante la determinación del porcentaje de inhibición del radical estable 1,1-difenil-2-picril-hidracilo. Los valores de la mediana de los consumidores respecto al grupo control fueron: volumen ­ 2,98 mL versus 3,95 mL (p = 0,0221); concentración total ­ 189 millones/mL versus 291,1 millones/mL (p = 0,0636); movilidad progresiva ­ 50% versus 56,5% (p = 0,0052); viabilidad ­ 65,3% versus 73,1% (p = 0,0732); y morfología normal ­ 5% versus 7% (p = 0,0167), respectivamente. Los resultados obtenidos en este estudio indican que el consumo de cigarrillos de marihuana afecta negativamente la movilidad progresiva, la morfología normal y la concentración total de espermatozoides; además, la concentración total de espermatozoides está afectada por la frecuencia del consumo de cigarrillos de marihuana.

Evidence suggests that exposure to psychoactive substances is related to spermatogenesis alterations that affect sperm quality. The objective of the present work was to determine sperm parameters in regular users of marijuana cigarettes. Seminal samples from 42 active consumers of marijuana cigarettes and 16 volunteer non-marijuana users were analyzed. Through a semen analysis, we identify conventional seminal parameters (viability, motility, morphology, and sperm concentration) according to the guidelines established by the World Health Organization (WHO). The antioxidant effect of the seminal plasma was evaluated through the determination of the percentage of inhibition of the stable radical 1,1-diphenyl-2-picrylhydrazyl. The median values of consumers with respect to the control group were respectively: volume ­ 2.98 mL versus 3.95 mL (p = 0.0221); total concentration ­ 189 million/mL versus 291.1 million/mL (p = 0.0636); progressive motility ­ 50% versus 56.5% (p = 0.0052); viability ­ 65.3% versus 73.1% (p = 0.0732); and normal morphology ­ 5% versus 7% (p = 0.0167). The results obtained in the present study indicate that the consumption of marijuana cigarettes negatively affects progressive motility, normal morphology, and total sperm concentration. In addition, the total sperm concentration is affected by the frequency of consumption of marijuana cigarettes

Up-regulation of Arl4a gene expression by broccoli aqueous extract is associated with improved spermatogenesis in mouse testes / La regulación positiva de la expresión del gen Arl4a por acción del extracto acuoso de brócoli se asocia con una mejor espermatogénesis en testículos de ratón

Abstract | Introduction: Broccoli (Brassica oleracea) is well known for its properties as an anticancer, antioxidant, and scavenger of free radicals. However, its benefits in enhancing spermatogenesis have not been well established. Objective: To study broccoli aqueous extract effects on sperm factors and the expression of genes Catsper1, Catsper2, Arl4a, Sox5, and Sox9 in sperm factors in mice. Materials and methods: Male mice were divided randomly into six groups: (1) Control; (2) cadmium (3 mg/kg of mouse body weight); (3) orally treated with 200 µl broccoli aqueous extract (1 g ml-1); (4) orally treated with 400 µl of broccoli aqueous extract; (5) orally treated with 200 broccoli aqueous extract plus cadmium, and (6) orally treated with 400 µl of broccoli aqueous extract plus cadmium. We analyzed the sperms factors and Catsper1, Catsper2, Arl4a, Sox5, and Sox9 gene expression. Results: An obvious improvement in sperm count and a slight enhancement in sperm motility were observed in mice treated with broccoli extract alone or with cadmium. Sperm viability was reduced by broccoli extract except for the 200 µl dose with cadmium, which significantly increased it. Interestingly, Arl4a gene expression increased in the 400 µl broccoli- treated group. Likewise, the Arl4a mRNA level in mice treated with cadmium and 200 µl of broccoli extract was higher than in the cadmium-treated mice. Furthermore, broccoli extract enhanced the mRNA level of Catsper2 and Sox5 genes in mice treated with 200 µl and 400 µl broccoli extract plus cadmium compared with the group treated solely with cadmium. Conclusion: The higher sperm count in broccoli-treated mice opens the way for the development of pharmaceutical products for infertile men.

Resumen | Introducción. El brócoli (Brassica oleracea) se conoce por sus propiedades como anticancerígeno, antioxidante y eliminador de radicales libres. Sin embargo, sus beneficios en la espermatogénesis aún no se han determinado suficientemente. Objetivo. Estudiar los efectos del extracto acuoso de brócoli sobre los factores espermáticos y la expresión de los genes Catsper1, Catsper2, Arl4a, Sox5 y Sox9 en ratones. Materiales y métodos. Los ratones machos se dividieron aleatoriamente en seis grupos: 1) control; 2) tratados con cadmio, 3 mg/kg de peso corporal; 3) tratados con 200 µl de extracto acuoso de brócoli (1 g ml-1); 4) tratados con 400 µl de extracto acuoso de brócoli; 5) tratados con 200 µl de extracto acuoso de brócoli más cadmio, y 6) tratados con 400 µl de extracto acuoso de brócoli más cadmio. El extracto acuoso de brócoli se administró por vía oral. Se analizaron los factores espermáticos y la expresión de los genes Catsper1, Catsper2, Arl4a, Sox5 y Sox9. Resultados. Se observó una mejoría obvia en el recuento y una ligera mejoría en la motilidad de los espermatozoides, en ratones tratados con extracto de brócoli solo o con cadmio. La viabilidad de los espermatozoides se redujo con el extracto de brócoli, excepto con la dosis de 200 µl más cadmio, la cual la aumentó significativamente. Curiosamente, la expresión del gen Arl4a aumentó en el grupo tratado con 400 µl del extracto. Asimismo, el ARNm del Arl4a en ratones tratados con cadmio y 200 µl del extracto, fue más abundante que en los ratones tratados solo con cadmio. Además, el extracto de brócoli aumentó la cantidad de ARNm de los genes Catsper2 y Sox5 en ratones tratados con 200 y 400 µl de extracto de brócoli más cadmio, en comparación con el grupo tratado únicamente con cadmio. Conclusión. El mayor número de espermatozoides en ratones tratados con brócoli abre el camino al desarrollo de productos farmacéuticos para hombres infértiles.

Scrotal thermographic profile and seminal characteristics of Mangalarga Marchador stallions bred in the Atlantic Rainforest biome / [Perfil termográfico escrotal e características seminais de garanhões Mangalarga Marchador criados no bioma Mata Atlântica]

The objective of this study was to determine the scrotal thermographic profile and to verify the influence of temperature and humidity of the humid tropical climate on testicular temperature and seminal quality of Mangalarga Marchador stallions. The thermal profiles of the proximal, middle, and distal zones of the testicles and total surface temperature (TSTT) were recorded using an FLIR E60bx thermal imager. The average air temperature (°C) and relative humidity (%) were obtained 1, 5, 9, 33, and 66 days before semen collection and showed a mean value of 26.5±2.4 and 80.4±6.0 respectively. The scrotal surface temperature was close to 34°C and there was no variation with the age of the stallion, reproductive activity, and characteristics of the ejaculate (P>0.05). The only significant correlations obtained were between TSTT and minor defects (R = 0.41; P<0.05), between TSTT and total defects (R = 0.46; P<0.01), and between TSTT and percentage of morphologically normal sperm (R = -0.46; P<0.05). It was concluded that the Mangalarga Marchador stallions maintained the testicular temperature within favorable conditions for spermatogenesis, demonstrating the efficiency of testicular thermoregulation mechanisms in the Atlantic Forest biome.

O objetivo deste estudo foi determinar o perfil termográfico escrotal e verificar a influência da temperatura e da umidade do clima tropical úmido na temperatura testicular e na qualidade seminal de garanhões Mangalarga Marchador. Os perfis térmicos das zonas proximal, média e distal dos testículos e a temperatura da superfície total (TSTT) foram registrados usando-se um termovisor FLIR E60bx. A temperatura média do ar (° C) e a umidade relativa (%) foram obtidas um, cinco, nove, 33 e 66 dias antes da coleta de sêmen e apresentaram valor médio de 26,5 ± 2,4 e 80,4 ± 6,0, respectivamente. A temperatura da superfície escrotal foi próxima a 34°C, e não houve variação com a idade do garanhão, a atividade reprodutiva e as características do ejaculado (P>0,05). As únicas correlações significativas obtidas foram entre TSTT e defeitos menores (R=0,41; P<0,05), entre TSTT e defeitos totais (R=0,46; P<0,01), e entre TSTT e porcentagem de espermatozoides morfologicamente normais (R=-0,46; P<0,05). Concluiu-se que os garanhões Mangalarga Marchador mantiveram a temperatura testicular dentro de condições favoráveis para a espermatogênese, demonstrando a eficiência dos mecanismos de termorregulação testicular no bioma Mata Atlântica.

Efecto de la diabetes sobre la espermatogénesis / Effect of diabetes on spermatogenesis

La evidencia clínica que ha permitido relacionar la diabetes mellitus con la infertilidad se basa en la importancia del metabolismo de la glucosa durante el proceso de espermatogénesis, debido a que en los episodios tanto de hipoglucemia como de hiperglucemia pueden ocurrir cambios epigenéticos en algunas proteínas involucradas en la espermatogénesis. En la presente comunicación se describen los aspectos teóricos de los efectos de la diabetes sobre el líquido seminal con énfasis en la espermatogénesis(AU)

The clinical evidence that has made it possible to link diabetes mellitus with infertility is based on the importance of glucose metabolism during the spermatogenesis process, because in episodes of both hypoglycemia and hyperglycemia, epigenetic changes can occur in some proteins involved in spermatogenesis. This communication describes the theoretical aspects of the effects of diabetes on seminal fluid with emphasis on spermatogenesis(AU)

Comparison of the spermatogenic process in three different mice strains: Swiss, Balb/C and C57BL/6

Many studies have been trying to establish standard protocols for animal experimentation, especially for animal species or strains, to master research variables with high precision. The main mouse strains used in the field of the biology of reproduction are Swiss, Balb/c, and C57BL/6. Since some of the strains show reproduction limitations, such as the size of the litter, the present study aimed to compare their spermatogenic processes to verify differences regarding the testicular parenchyma and germ cell populations, which could explain low offspring production. In addition, the present study provides additional information concerning the testicular parenchyma of such strains, which consequently would help researchers to choose the most suitable strain for reproductive studies. Six adult male mice were used for each of the strains. After euthanasia, the testes were weighed, fixated with Karnovsky fixative, embedded in methacrylate, sectioned, and stained with toluidine blue/sodium borate 1%. Morphometrical analyses from the testicular parenchyma (seminiferous tubules and interstitium) were made using the software ImageJ. Germ and Sertoli cells populations were counted in seminiferous tubules cross-sections at stage I of the seminiferous epithelium cycle. The lowest body and testicular weights were observed in C57BL/6 animals, followed by Balb/c and Swiss, however, the relative testes, parenchyma, and albuginea weights were significantly lower only in C57BL/6. Despite the seminiferous tubules and seminiferous epithelium proportions were lower in Swiss animals, their relative amount related to the body weight was the same among strains. The total number of germ cells was higher in Swiss animals, reflecting higher spermatogenic yield and daily sperm production. Due to the lower relative number of Sertoli cells, the Swiss animals showed the highest Sertoli cell index and support capacity. On the other hand, the lowest pathological indexes regarding the germ cells were observed in Balb/c animals, followed by Swiss and C57BL/6. In the interstitium, the proportion of blood vessels was lower in Swiss mice, while the lymphatic cell proportion was lower in C57BL/6 animals. Moreover, the highest proportions of Leydig cells and macrophages were noticed in Swiss mice, which may indicate increased testosterone levels. Altogether, such observations must be taken into account when choosing any of the studied strains for reproduction studies.