Lymphomas affecting the submandibular glands.

BACKGROUND: Lymphomas affecting the submandibular glands are very uncommon and few reports are currently available in the literature. Therefore, the aim of the current study is to describe the clinical and microscopic features of an original series of lymphomas affecting the submandibular glands. MATERIAL AND METHODS: The pathology files of two institutions were searched for lymphoma cases affecting the submandibular glands. The original hematoxylin and eosin, and immunohistochemical slides were revised by a pathologist for diagnosis confirmation following the revised 4th edition of the World Health Organization classification of tumours of haematopoietic and lymphoid tissues. Clinical data regarding age, sex, clinical manifestation, treatment, follow-up and status at last appointment were retrieved from the patients' medical charts. RESULTS: During the period investigated, 16 cases were included in the study. Females predominated (10:6) with a mean age of 57.8 years-old. Tumors usually presented as asymptomatic swellings. MALT lymphoma represented the most common subtype, followed by diffuse large B cell lymphoma and follicular lymphoma. Three patients died, one of them affected by plasmablastic lymphoma, one by DLBCL and one by MALT lymphoma. CONCLUSIONS: Low-grade B cell lymphomas predominate in the submandibular glands, but DLBCL and other subtypes may also be rarely diagnosed in this salivary gland.

Transcriptional downregulation of ABC transporters is related to follicular degeneration after vitrification and in vitro culture of ovine ovarian tissue.

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.

In vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-µg/mL insulin and 100-µg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 µm.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro.

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50ng mL-1 AMH and/or 100ng mL-1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0-9) and second (Days 10-18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH+FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P<0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P>0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.

Fibrin-alginate hydrogel supports steroidogenesis, in vitro maturation of oocytes and parthenotes production from caprine preantral follicles cultured in group.

This study aimed to establish a culture system that improves the in vitro development of caprine preantral follicles. In a first experiment, follicles were encapsulated as a single unit per bead and cultured singly or in groups or with five follicles in the same alginate (ALG) bead for 18 days. In a subsequent experiment, the "five follicles per bead" design was chosen to culture in ALG, fibrin-alginate (FA) or hyaluronate (HA) for 18 days. In a third experiment, we chose the five follicles per bead in FA to culture for 30 days. The culture set-up of five follicles per ALG bead increased antrum formation and follicle diameter compared to the other culture designs (p < .05). Moreover, under this condition, 44.44% of the oocytes from in vitro cultured preantral follicles reached meiotic resumption. A significant increase of follicle diameter occurred in attachment system and FA (p < .05), but the ALG condition reached the highest among all groups on day 18 (p < .05). Follicles encapsulated in matrix produced more estradiol and progesterone than attachment system (p < .05). The expression of MMP-9 mRNA was higher in FA than in other groups (p < .05) and similar to antral follicles from in vivo control (p > .05). Only FA group resulted in oocytes matured. After 30 days, oocytes from preantral follicles in vitro grown in FA developed to eight-cell parthenotes. In conclusion, a culture system using FA supported the development of caprine preantral follicles cultured in group and included in the same bead of hydrogel, improving the oocyte maturation and producing parthenotes.

Fotoperíodo e frequência alimentar na larvicultura do peixe beta / Photoperiod and feeding frequency in the Siamese fighting fish hatchery

Com este trabalho, objetivou-se avaliar o crescimento, a uniformidade e a sobrevivência das larvas de Betta splendens, submetidas a diferentes fotoperíodos e frequências de alimentação. Foram distribuídos aleatoreamente 480 indivíduos (4,53mg ± 0,32 e 5,51 ± 0,58mm) em 48 recipientes plásticos (1L), com densidade de 10 larvas/ L. Foi utilizado um delineamento experimental inteiramente ao acaso, com quatro repetições, em arranjo fatorial 6x2, com seis fotoperíodos (0L:24E, 6L:18E, 12L:12E, 16L:8E, 20L:4E, 24L:0E) e duas frequências de alimentação (duas ou quatro vezes/ dia). Durante um período de 15 dias, as larvas foram alimentadas com náuplios de Artemia, na proporção de 800 náuplios/ larva/ dia. Larvas de beta submetidas aos fotoperíodos de 12L:12E e 16L:8E apresentaram o maior crescimento em peso (P<0,10), enquanto as que foram alimentadas quatro vezes ao dia apresentaram maior crescimento em comprimento e uniformidade (P<0,10). No entanto, os indivíduos que foram alimentados quatro vezes ao dia apresentaram menor sobrevivência quando submetidos aos fotoperíodos de 16L:8E, 20L:4E e 24L:0E (P<0,10). Por outro lado, as larvas submetidas aos fotoperíodos de 12L:12E, 16L:8E e 20L:4E apresentaram maior taxa de sobrevivência quando alimentadas duas vezes ao dia (P<0,10). Portanto, ao se preconizar maior crescimento, uniformidade e sobrevivência das larvas de Betta splendens, recomenda-se a realização da larvicultura dessa espécie sob o fotoperíodo de 12L:12E, com o fornecimento de náuplios de Artemia em duas alimentações diárias.(AU)

The aim of this study was to evaluate the growth, uniformity and survival of Betta splendens larvae, submitted to different photoperiods and feeding frequency. Four hundred and eighty individuals (4.53mg ± 0.32 and 5.51 ± 0.58mm) were randomly distributed into 48 plastic containers (1L) at a density of 10 larvae/L. A completely randomized design was used, with four replications in a factorial 6 x 2, six photoperiods (0L:24D, 6L:18D, 12L:12D, 16L:8D, 20L:4D, 24L:0D) and two feeding frequencies (two or four times a day). The larvae were fed performed with Artemia nauplii at averaging 800/ larvae/ day, for 15 days. Beta larvae subjected to photoperiod 12L:12D and 16L:8E showed the greatest weight gain (P<0.10), while those fed four times daily had greater length growth and uniformity (P<0.10). However, individuals fed four times daily had lower survival when subjected to photoperiod 16L:8E, 20L:4L and 24E:0D (P<0.10). On the other hand, larvae subjected to a photoperiod of 12L:12D, 16L:8L and 20L:4E showed higher survival rate when fed twice a day (P<0.10). Therefore, with the intention of better growth, uniformity and survival of Betta splendens larvae, it is recommended that the hatchery in this species be done under a photoperiod of 12L: 12D with supply of Artemia nauplii twice daily.(AU)

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.

Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture.

Our aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P < 0.05) of AQP3 mRNA. The mRNA transcript levels were reduced (P < 0.05) for AQP9 in tissue following er-EG+DMSO. Immunolocalization was positive for both proteins (AQP3 and AQP9) on ovine preantral follicles following all treatments, except in the e-EG+DMSO group. In Experiment II, the mRNA levels of AQP3 and AQP9 following VIT treatment were similar (P > 0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality.

Steady-state level of messenger RNA and immunolocalization of aquaporins 3, 7, and 9 during in vitro growth of ovine preantral follicles.

Aquaporins (AQPs) are a well-conserved family of small (approximately 30 kDa) membrane channel proteins that facilitate rapid movement of fluids and have a unique tissue-specific pattern of expression. These proteins have been found in the female reproductive systems of humans, rats, and mice. However, the expression and cellular localization of AQPs have not extensively been studied in the female reproductive system of sheep. Therefore, this study aimed to evaluate, by real-time polymerase chain reaction and immunohistochemistry respectively, the levels of messenger RNA and the immunolocalization of AQP3, AQP7, and AQP9 in large isolated ovine secondary follicles over a period of IVC. Our analysis revealed that AQP3 and AQP9 were present predominately in follicles that exhibited antrum formation, suggesting a crucial role of these AQPs in the formation of the antrum. Interestingly, AQP7 was only expressed in follicles that had not formed an antrum by Day 12 of culture. In conclusion, the presence of protein channels (AQP3 and AQP9) seems to be essential for the formation of the antrum in isolated ovine secondary follicles cultured in vitro and thus plays an important role during folliculogenesis in this species.

Differential gene expression and immunolocalization of platelet-derived growth factors and their receptors in caprine ovaries.

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-ß were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-ß (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-ß were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats.

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-ß) and their effect on in vitro development of caprine preantral follicles.

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-ß) and its receptors (TGF-ßRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-ß, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-ß and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-ß receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-ß (10 ng/ml), or TGF-ß + FSH for 18 d. TGF-ß increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-ß in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-ß and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.

Alginate hydrogel matrix stiffness influences the in vitro development of caprine preantral follicles.

This study examined caprine follicular development in different concentrations of alginate matrix to determine the optimal conditions for culture. Caprine preantral follicles were cultured in a two-dimensional system (control) or a three-dimensional encapsulated system in 0.25%, 0.5%, or 1% alginate (ALG 0.25, ALG 0.5, and ALG 1, respectively). A higher percentage of morphologically normal follicles developed in ALG 0.5 and ALG 1 than in ALG 0.25 or the control (P < 0.05). The rate of antrum formation, however, was higher in ALG 0.25 than in ALG 0.5 and ALG 1 conditions (P < 0.05), but similar to the control. Follicles cultured in ALG 0.25 had higher growth rates and meiotic resumption than those cultured in ALG 0.5, ALG 1, or the control (P < 0.05). Moreover, follicles cultured in ALG 0.25 had higher levels of estradiol and progesterone than those cultured in ALG 0.5, ALG 1, or the control, as well as higher levels of CYP19A1 and HSD3B mRNA. In conclusion, a three-dimensional system that uses ALG 0.25 fosters the in vitro development of caprine preantral follicles and increases the rate of meiotic resumption.

Structure, function, and localization of aquaporins: their possible implications on gamete cryopreservation.

The discovery of water channels (aquaporins, AQPs) was a landmark event for the clarification of water transport through the plasma membrane. AQPs belong to a family of intrinsic membrane proteins that act as selective channels for water and for solutes such as glycerol and urea. AQPs were found in different tissues and organs, including male and female reproductive systems. In the swine female reproductive system, the AQPs were localized in the uterus, oviduct, and ovary, as well as in the granulosa cells from primordial follicles. Knowing the involvement of AQPs with the male and female germ cells, as well as their acknowledged role in transporting water through the plasma membrane, the research of these proteins in cryopreservation processes becomes essential. Thus, this review aims to describe the structure and function of AQPs in membranes, highlighting their role in the reproductive system (male and female). We also discuss the involvement of AQPs in cryopreservation, focusing on the effect and importance of these proteins on the rates of vitrification protocols for preantral follicles present in the ovarian tissue of domestic mammals.

Shoulder pathology associated with symptomatic acromioclavicular joint degeneration.

We report the incidence and nature of shoulder disease found in association with symptomatic degenerative change in the acromioclavicular joint in 218 shoulders. Coexisting pathologic conditions were present in 213 shoulders: rotator cuff degeneration in 176 shoulders (79 with complete thickness tears), labral tears in 72, glenohumeral degeneration in 31, and biceps tendon disease in 49. In 59 shoulders findings were unexpected. We looked specifically at 2 age groups: <50 years and > or =50 years. In the <50 years group labral tears were seen in 42% (30 of 71) and rotator cuff disease in 65% (46 of 71). Cuff tears tended to be incomplete. In the older age group only 14% (21 of 147) had an intact cuff, with 72 shoulders having a full-thickness tear. Labral tears were seen in 29% (42 of 147). We recommend that all patients undergo shoulder arthroscopy at the time of acromioclavicular surgery.