Maternal chronic caffeine intake impairs fertility, placental vascularization and fetal development in mice.

Caffeine is commonly consumed by pregnant women to avoid fatigue or as a habit. However, it is not clearly determined its side effects to the conceptuses. This study evaluated placental morphofunctional alterations after maternal chronic caffeine intake and the effects on fetal growth. Female Swiss mice received, via gavage, caffeine doses (either 60, 120 or 240 mg/kg/day) seven days before mating until gestational days-(GD) 11.5 or 17.5. Fetal biometrical parameters were assessed, and placentae were either submitted to histomorphometrical or molecular evaluation of angiogenesis (placental growth factor-1[PlGF-1]), apoptosis (Caspase-3) and proliferation (Ki-67) markers (evaluated in Swiss dams) and to intravital microscopy (evaluated in C57BL/6 dams). Caffeine exposed fetuses exhibited intrauterine growth restriction in a sex-dependent manner, with greater commitment of female fetuses (P < 0.05). In addition, placentae from dams that received 120 mg/kg/day showed less irrigation by maternal blood and greater development of fetal vasculature, characterized by higher number of larger vessels (P < 0.05). Although no effects on apoptosis (Caspase-3) and angiogenesis (PlGF-1) were observed, dams treated with 60 mg/kg/day showed greater placental cell proliferation (Ki-67 staining) at GD 11.5 (P < 0.05). The group treated with 240 mg/kg/day exhibited only one pregnant dam for each gestational age, suggesting that this high caffeine consumption may compromise fertility. Taken together, even in the doses currently ingested by many pregnant women, caffeine has detrimental effects on placental vasculature and fetal development in mice. Therefore, our results strongly suggest that caffeine consumption in human pregnancies greater than the recommended doses should be avoided.

Optimization of Testicular Fixation-Embedding Techniques for Improved Evaluation of Mammalian Spermatogonial Morphology and Function.

Numerous methods have been successfully used to evaluate mammalian spermatogonial biology However, the conventional light microscopy assays present a challenge in precisely identifying spermatogonial phenotypes, which can result in discrepancies between molecular and morphological findings. Such precise association could lead to a more robust interpretation of spermatogonial activity in steady-state spermatogenesis, which may facilitate the translation from basic research to clinical applications. In this chapter, we present two histological processing methods that enable a comprehensive analysis of spermatogonial morphology and function, involving fixation of mammalian testicular tissue in glutaraldehyde and embedding in plastic resin. These techniques have proven to be effective in light microscopy studies.

High SARS-CoV-2 tropism and activation of immune cells in the testes of non-vaccinated deceased COVID-19 patients.

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

Intrauterine growth restriction and its impact on intestinal morphophysiology throughout postnatal development in pigs.

Intrauterine growth restriction (IUGR) compromises fetal development, leading to low birth weight, and predisposes to gastrointestinal disorders. Pigs that suffered IUGR present poor postnatal development, resulting in great economic losses to the industry. The small intestine may be involved with impaired development, but studies investigating this issue are still limited. Thus, the present study aimed to investigate small intestine morphofunctional alterations in IUGR pigs throughout the production phases (birth to 150 days). IUGR pigs presented lower body weight from birth to the finishing phase (P < 0.05). Although histomorphometrical parameters were not affected during the pre-weaning period, their commitment was observed specifically in the duodenum of the IUGR group at older ages (P < 0.05). The most detrimental effects on the small intestine, such as deeper duodenum crypts' depth, lower villus height:crypt depth ratio and absorptive area, increased apoptosis and lower proliferation of the duodenum epithelium were noticed at 70 days of age (P < 0.05). Additionally, IUGR pigs presented the lowest chymotrypsin and amylase activities at 70 and 150 days of age, respectively (P < 0.05). These findings may contribute to the elucidation of morphofunctional disorders of the small intestine in IUGR pigs throughout the different production phases, suggesting that poor postnatal development may be due to intestinal damage.

Birthweight leads to seminal and testicular morphofunctional commitment in sexually mature boars.

Given the importance of males as semen donors for artificial insemination (AI) and the high incidence of low birthweight piglets at commercial farms, the impact of birthweight on fertility in boars deserves special attention. The aim of this study was to evaluate testicular morphofunctional parameters and semen characteristics in different birthweight boars. Forty littermate males were selected at birth and divided into two experimental groups, according to birthweight: high (HW, birthweight ranging from 1.80 to 2.15 kg, n = 20) and low (LW, birthweight ranging from 0.75 to 1.10 kg, n = 20). At 170 days of age, a sub-set of 24 littermate boars (n = 12 HW and n = 12 LW) were randomly selected for semen collection, which was performed once a week, at a 15-day interval, during five weeks. At 300 days of age, boars were orchiectomized, and the testis processed for histological and molecular analyses. The HW group was heavier and presented larger testes compared to LW animals (P < 0.05). Despite that, birthweight did not significantly affect semen volume or sperm quality parameters (concentration, motility, vigor or morphology), although LW boars produced 38.2% fewer total sperm and 24% lower semen concentration, leading to 36.8% less inseminating doses. The histomorphometrical evaluation showed that seminiferous tubules diameter and germinal epithelium height were similar between experimental groups. However, LW boars presented shorter seminiferous tubules and, consequently, fewer Sertoli cells per testis (P 0.05). Even though plasma testosterone levels were equivalent in both birthweight groups, LW testis presented less androgen receptors (P < 0.05). Additionally, birthweight was positively correlated with total seminiferous tubule length and number of Sertoli cells (P < 0.01), and with body and testis weights (P < 0.01). Taken together, even though adult LW boars showed no evident semen pathologies or spermatogenesis commitment, mature HW males have the potential to produce more spermatozoa, consequently more semen doses per ejaculate, being more valuable to an industry that relies on AI.

Comparison between the techniques of inclusion in glycol methacrylate (GMA)-based plastic resin and paraffin for evaluation intestinal morphometry in horses.

To determine the effect of the inclusion method on the histomorphometric evaluation of the gastrointestinal mucosa of horses, jejunum samples were collected using flank laparotomy. Sixteen mixed breed healthy adult horses, including four males and 12 females, aged 4-14 years with an average body weight of 248.40 ± 2.28 kg, were used. Jejunal biopsies were collected and analyzed by light microscopy using two methods: group 1 comprised biopsies fixed using 10% neutral formalin and embedded in paraffin; biopsies in group 2 were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2, followed by inclusion in glycol methacrylate (GMA)-based plastic resin. Intestinal villi height, crypt depth, glandular mucosa thickness, total mucosal thickness, and villus/crypt ratio were then evaluated. For all the variables studied, with exception of the villus/crypt ratio, a significant difference (p < 0.05) was found between samples in groups 1 and 2. Processing samples for embedding in plastic resin was quicker and easier to perform compared to that for paraffin embedding. In addition, the epithelial lining of tissues in group 2 showed better resolution for conducting cytological studies under a light microscope. The difference between the studied variables can be attributed to tissue retraction caused by conventional processing for inclusion in paraffin. Therefore, the method of inclusion in GMA described in the present study appears to be a more reliable choice for morphometric evaluation of the intestinal mucosa of horses.

Para determinar o efeito do método de inclusão sobre a avaliação histomorfométrica da mucosa gastrointestinal de equinos foram coletadas amostras do jejuno por laparotomia pelo flanco. Foram utilizados 16 equinos adultos hígidos, sem raça definida, de ambos os sexos, quatro machos e 12 fêmeas, com idade variando entre quatro e 14 anos, e peso corporal médio de 248,40 + 2,28kg. Amostras do jejuno foram coletadas e processadas para biopsia em microscopia de luz sob dois métodos: grupo 1 - fixação em formalina neutra tamponada a 10% e inclusão em parafina, grupo 2 - fixação em glutaraldeído 2,5% em tampão fosfato 0,1M pH 7,2, seguido de inclusão em resina plástica à base de glicol metacrilato. Os seguintes parâmetros foram avaliados: altura das vilosidades intestinais, profundidade de cripta, espessura da mucosa glandular, espessura total da mucosa e relação vilo/cripta. Para todas as variáveis estudadas, exceto relação vilo/cripta, foi encontrada diferença significativa (p<0,05) entre os dois métodos. O processamento para inclusão em resina plástica foi rápido e de fácil execução quando comparado à inclusão em parafina. Além disso, o epitélio de revestimento apresentou melhor resolução das células para estudo histológico ao microscópio de luz. A diferença entre as variáveis pode ser atribuída a retração do tecido provocada pelo processamento convencional por inclusão em parafina. Portanto, o método de inclusão em GMA mostrou-se, no presente estudo, uma escolha mais fidedigna para as avaliações morfométricas da mucosa intestinal de equinos.

Maternal protein restriction before and during pregnancy leads to a gestational day-dependent response of folliculogenesis in outbred mice.

Knowledge of follicle development during pregnancy under experimental conditions could be a key factor to understanding maternal ovarian activity. Thus, this study evaluated the effects of maternal protein restriction before and during pregnancy on folliculogenesis. Swiss outbred female mice were allocated to either a control (CC; 20% protein) or treated (TT; 8% protein) group. Pregnant females were killed either on Gestational day (GD) 7.5 or GD17.5 and the ovaries were evaluated using histomorphometric and immunohistochemical methods. TT females showed higher feed and energy intakes, but lower bodyweight gain at GD17.5 (P<0.05). They also had lower number of secondary follicles at GD7.5 and a higher proportion of primordial follicles at GD17.5 (P<0.05). In addition, the areas of the secondary follicles and their granulosa layer were smaller in the TT group on GD7.5, whereas the areas of the oocyte and granulosa layer from atretic follicles were larger (P<0.05). Notwithstanding the slight increase in the insulin-like growth factor 1 (IGF1) receptor expression on GD7.5 in the TT group, there was a marked reduction in IGF1 expression detected in secondary follicles on GD17.5 (P<0.05). Collectively, these results demonstrate that protein restriction during pregnancy negatively affects follicle quality by reducing the size and activation capacity, which is more severe in late pregnancy.

Ovarian morphometrical evaluation to assess reproductive activity suppression in heavy weight finishing gilts immunized against gonadotropin-releasing hormone.

It is known that immunizing gilts against gonadotropin-releasing hormone (GnRH) is an efficient castration method that increases their growth performance. However, it is still unknown the ovarian histophysiology outcomes after this procedure. Therefore, the aim of this study was to investigate in detail, using morphological and morphometrical methods, changes in the ovarian structure that result in the suppression of ovarian activity, as well as to gain knowledge on the ovarian structure to assist in ovarian histopathological diagnoses. Seventy-two pre-pubertal finishing gilts were allocated to two experimental groups: immunized (IC; n = 36; gilts which received two injections of 2 mL of Vivax® - one at 15 and another at 19 weeks of age) and control (CT; n = 36, females which received two saline injections following the same protocol). All gilts were euthanized at 25 weeks of age and the ovaries of 5 gilts from each experimental group collected for biometrical and histomorphometrical analysis. IC gilts showed higher body weights, but smaller ovaries compared to CT females. In addition, the number of small follicles (≤ 2 mm) on the ovarian surface was higher, while no large follicles (> 6 mm) nor corpora lutea were found in the ovaries of IC gilts. Histomorphometrical analysis revealed that IC females showed higher numbers of quiescent and active primordial, primary, pre-antral and final stage atretic follicles. Moreover, follicle size, antrum diameter and area of the granulosa layer from mature follicles were smaller in IC gilts. Collectively, these results demonstrate that the efficacy of immunization against GnRH is related to the blockage of follicular recruitment and selection, thus suppressing reproductive activity in finishing gilts.

A fertility-oriented method for histological processing of testicular biopsies in men with azoospermia.

The present study was designed to evaluate whether tissue preparation by glutaraldehyde and glycol methacrylate (G/GMA) improves the diagnostic assessment of testicular biopsies from azoospermic men when compared to the standard tissue preparation using Bouin's solution and paraffin. We prospectively included a total of 21 testicular biopsies of sexually mature men aged 29-50 years with infertility and azoospermia. One testicular biopsy fragment from each patient was processed by the G/GMA method, whereas another tissue fragment was contemporarily processed by the conventional Bouin/paraffin (B/P) method. The G/GMA method provided better resolution of cytological details of the seminiferous epithelium, changing the final diagnosis in four cases. The medians of Bergmann's spermatogenesis scores were 0.25 (interquartile range 0.04-0.88) for B/P preparations and 0.79 (interquartile range 0.17-0.96) for G/GMA preparations. Both techniques allowed accurate prediction of sperm recovery from the biopsies (B/P, area under the receiver operating characteristics [ROC] curve 0.88, 95% confidence interval [CI] 0.75-1.00; G/GMA, area under the ROC curve 0.94, 95% CI 0.86-1.00). We conclude that human testicular biopsy preparation with G/GMA improved image resolution under light microscopy and produced more reliable results for the evaluation of spermatogenesis in comparison with B/P, allowing a more precise fertility-oriented diagnosis in azoospermic men.Abbreviations: B/P: Bouin/paraffin; GMA: glycol methacrylate; G/GMA: glutaraldehyde and glycol methacrylate; ICSI: intracytoplasmic sperm injection; OA: obstructive azoospermia; NOA: nonobstructive azoospermia; TESE: testicular sperm extraction.

Characterization of neoplastic cells outlining the cystic space of invasive micropapillary carcinoma of the canine mammary gland.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a rare malignant breast tumor and a variant form of invasive ductal carcinoma that is an aggressive neoplasm of the human breast and canine mammary gland. The importance of the tumor microenvironment in cancer development has gradually been recognized, but little is known about the cell types outlining the cystic space of canine IMPC. This study aimed to characterize the neoplastic cells outlining the cystic space of IMPC. RESULTS: Immunohistochemistry (IHC), immunofluorescence (IF), superresolution and transmission electron microscopy (TEM) were used to assess the cell types in the cystic areas of IMPCs. Cells expressing the mesenchymal markers alpha-smooth muscle actin (αSMA), Vimentin, and S100A4 outlined the cystic space of IMPC. Furthermore, loss of epithelial cell polarity in IMPC was shown by the localization of MUC1 at the stroma-facing surface. This protein modulates lumen formation and inhibits the cell-stroma interaction. Immunohistochemical and IF staining for the myoepithelial cell marker p63 were negative in IMPC samples. Furthermore, associated with peculiar morphology, such as thin cytoplasmic extensions outlining cystic spaces, was observed under TEM. These observations suggested cells with characteristics of myoepithelial-like cells. CONCLUSIONS: The cells outlining the cystic space of IMPC in the canine mammary gland were characterized using IHC, IF and TEM. The presence of cells expressing αSMA, Vimentin, and S100A4 in the IMPC stroma suggested a role for tumor-associated fibroblasts in the IMPC microenvironment. The reversal of cell polarity revealed by the limited basal localization of MUC1 may be an important factor contributing to the invasiveness of IMPC. For the first time, the cystic space of canine mammary gland IMPC was shown to be delimited by myoepithelial-like cells that had lost p63 expression. These findings may enhance our understanding of the cellular microenvironment of invasive tumors to improve cancer diagnosis and treatment.