Osteocalcin improves testicular morphology but does not ameliorate testosterone synthesis signaling in azoospermic mice.

Objective: Osteocalcin (OCN) influences spermatogenesis in conjunction with testosterone and estrogen. OCN facilitates the secretion of testosterone by engaging with G protein-coupled receptor class C group 6 member A (GPRC6A) on Leydig cells and with androgen receptors on Sertoli cells. Methods: Adult mice were assigned to the following groups: control; sham I, which received dimethyl sulfoxide for 5 weeks followed by phosphate-buffered saline for 1 month; azoospermia, which was treated with busulfan (40 mg/kg); sham II, which consisted of azoospermic animals that received phosphate-buffered saline for 1 month beginning at the 5-week mark; and the experimental group, which included azoospermic mice treated with OCN (3 ng/g/day) for 1 month. Results: In the mice receiving OCN treatment, immunohistochemical analysis revealed increased expression of androgen receptors and GPRC6A, indicative of enhanced spermatogenesis. Additionally, the expression levels of the cyclic adenosine monophosphate-responsive element binding protein 1, steroidogenic acute regulatory protein, and cytochrome P450 family 11 genes were elevated. However, testosterone levels exhibited no significant differences across groups. Morphometric analysis suggests that OCN may play a crucial role in spermatogenesis, as evidenced by its positive effects on germinal cells and the germinal epithelium in the azoospermia group (p<0.05). Conclusion: We conclude that OCN may serve as a beneficial therapeutic agent for male infertility.

The effect of human menstrual blood-derived stem cells on ovarian folliculogenesis, angiogenesis and collagen volume in female rats affected by the polycystic ovary syndrome.

BACKGROUND: Infertility is one of the common problems among couples, affecting millions of people worldwide. Polycystic ovary syndrome (PCOS) is one of the main causes of infertility in women and is associated with abnormal folliculogenesis, angiogenesis and fibrosis. Common treatments may lead to numerous adverse effects on the patient's quality of life. The present study aimed to investigate the effects of human menstrual blood-derived stem cells on the ovarian histology of a PCOS model of Wistar rats. RESULTS: Based on the Papanicolaou test and H&E staining results, the number of primary, secondary and antral follicles in the PCOS and PCOS-Sham groups significantly increased compared to the control group, while they significantly decreased in the PCOS + Stem cells group compared to the PCOS and PCOS-Sham groups. Further, the number of atretic follicles in both PCOS and PCOS-Sham groups significantly increased in comparison with the control group and decreased in the PCOS + Stem cells group, compared to the two mentioned groups. Moreover, the Graafian follicles number was decreased in the PCOS and PCOS-Sham groups to significantly increase in the PCOS + Stem cells group. Based on Masson's trichrome staining, the number of blood vessels in PCOS and PCOS-Sham groups significantly increased compared to the control group, while a decrease was observed in the PCOS + Stem cells group, compared to PCOS and PCOS-Sham groups. CONCLUSION: The administration of MenSCs improved folliculogenesis in rats with polycystic ovaries. Also, MenSCs could ameliorate PCOS symptoms by improving fibrosis as well as angiogenesis and weight gain.

Application of Tissue-Specific Extracellular Matrix in Tissue Engineering: Focus on Male Fertility Preservation.

In vitro spermatogenesis and xenotransplantation of the immature testicular tissues (ITT) are the experimental approaches that have been developed for creating seminiferous tubules-like functional structures in vitro and keeping the integrity of the ITTs in vivo, respectively. These strategies are rapidly developing in response to the growing prevalence of infertility in adolescent boys undergoing cancer treatment, by the logic that there is no sperm cryopreservation option for them. Recently, with the advances made in the field of tissue engineering and biomaterials, these methods have achieved promising results for fertility preservation. Due to the importance of extracellular matrix for the formation of vascular bed around the grafted ITTs and also the creation of spatial arrangements between Sertoli cells and germ cells, today it is clear that the scaffold plays a very important role in the success of these methods. Decellularized extracellular matrix (dECM) as a biocompatible, functionally graded, and biodegradable scaffold with having tissue-specific components and growth factors can support reorganization and physiologic processes of originated cells. This review discusses the common protocols for the tissue decellularization, sterilization, and hydrogel formation of the decellularized and lyophilized tissues as well as in vitro and in vivo studies on the use of the testis-derived dECM for testicular organoids.

Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model.

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion-a mouse model for long-term infertility-was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C-Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6-integrin, and ß1-integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.

Orbital Lateral Cantal Distance Can Predict the Cephalometric Characteristic of Mandible in Iranian Population.

ABSTRACT: Orbitofacial anthropometrics have become an important tool used in reconstructive surgery. The authors attempt to evaluate the relation between orbital lateral canthal distance and the cephalometric characteristic of mandible in Iranian population.In a cross-sectional study, anthropometric parameters of face in 200 subjects (100 males and 100 females) with mean age of 34.39 ±â€Š18.83 were evaluated by three-dimensional computed tomography imaging.In this study, there was not a significant difference in the age of sex groups (P = 0.183). Also, there was no significant difference in the left and right mandible angle in different sex groups (P = 0.25, P  =  0.124, respectively). There were significant differences in the anterior mandible distance, inferior mandible angle distance (P = 0.0001) and lateral cantus distance of sex groups (P = 0.0001). There was a significant correlation between lateral contuse distance and left mandible angle (r = 0.226, P = 0.001), right mandible angle (r = 0.283, P = 0.00), mandible angle (r = -0.266, P = 0.00), anterior mandible angle distance (r = 0.655, P = 0.00), and inferior mandible angle distance (r = 0.582, P = 0.00).Here, we conclude that orbital lateral canthal distance can predict the cephalometric characteristic of mandible in Iranian population.

Pentoxifylline improves the survival of spermatogenic cells via oxidative stress suppression and upregulation of PI3K/AKT pathway in mouse model of testicular torsion-detorsion.

Testicular torsion-detorsion results in enhanced formation of free radicals which contribute to the pathophysiology of testicular tissue damage. Recent reports have identified protective role of pentoxifylline (PTX) against free radicals. Thus, we determined the protective effect of pentoxifylline against testicular damage in mouse model of testicular torsion-detorsion. Twenty (6 weeks old) male mice were divided into 4 groups of 5 animals each namely: Control (sham operated group), T1 (Torsion-detosion + single dose 100 mg/kg PTX, T2 (torsion-detorsion + 20 mg/kg PTX for 2 weeks and T/D (torsion-detorsion only). Animals in T1, T2 and T/D groups underwent 2 h of testicular torsion with the left testes rotated 720° (clockwisely) followed by 30 min of detorsion. After detorsion, drug administration was done intraperitoneally. The left testes of all the animals were excised on the 35th day after torsion-detortion for histopathological and biochemical assay. Histomorphological analysis of the seminiferous tubules showed that there were significant increase (P < 0.01 or 0.05) in the mean seminiferous tubule diameter, Johnson score and germ cells of animals in Control and T1 compared to T2 and T/D with no significant difference (P > 0.05) in testes weight, sertoli, leydig and myoid cells in all groups. IHC results showed significant increase (P < 0.01 or 0.05) in id4 and scp3 protein markers in Control, T1 and T2 compared to T/D. Oxidative stress analysis revealed that Pentoxifylline significantly increased (P < 0.01 or 0.05) the level of SOD, catalase, mRNA expression of akt and pi3k genes but significantly suppress (P < 0.01 or 0.05) MDA and Caspase-3 level in Control, T1 and T2 compared to T/D. Pentoxifylline could be used as an adjunct therapy to surgery in the treatment of torsion-detorsion related testicular injury, However, Further studies are needed to evaluate the effects of pentoxifylline on testicular torsion.

Roles for Kisspeptin in proliferation and differentiation of spermatogonial cells isolated from mice offspring when the cells are cocultured with somatic cells.

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10-8 -10-6 M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP3 , TP1, TP2 , and, Prm1 markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis.

Roles for osteocalcin in proliferation and differentiation of spermatogonial cells cocultured with somatic cells.

Spermatogonial cells (SCs) are key cells for spermatogenesis. These cells are affected by paracrine signals originated from nearby somatic cells, among them Leydig cells have receptors for osteocalcin, a hormone known for exerting positive roles in the promotion of spermatogenesis. The aim of this study was to evaluate roles for osteocalcin on SCs proliferative and differentiation features after coculture with Leydig cells. SCs and Leydig cells were isolated from neonate NMRI offspring mice and adult NMRI mice, respectively. SCs population were then enriched in a differential attachment technique and assessed for morphological features and identity. Then, SCs were cocultured with Leydig cells and incubated with osteocalcin for 4 weeks. Evaluation of proliferation and differentiation-related factors were surveyed using immunocytochemistry (ICC), Western blot, and quantitative real-time polymerase chain reaction (PCR). Finally, the rate of testosterone release to the culture media was measured at the end of 4th week. Morphological and flow cytometry results showed that the SCs were the population of cells able to form colonies and to express ID4, α6-, and ß1-integrin markers, respectively. Leydig cells were also able to express Gprc6α as a specific marker for the cells. Incubation of SCs/Leydig coculture with osteocalcin has resulted in an increase in the rate of expressions for differentiation-related markers. Levels of testosterone in the culture media of SCs/Leydig was positively influenced by osteocalcin. It could be concluded that osteocalcin acts as a positive inducer of SCs in coculture with Leydig cells probably through stimulation of testosterone release from Leydig cells and associated signaling.

Inductive role of sustentacular cells (Sertoli cells) conditioned medium on bone marrow derived mesenchymal stem cells / Papel inductivo del medio acondicionado de células sustentaculares (células de Sertoli) en las células madre mesenquimales derivadas de la médula ósea

RESUMEN: Las células madre de la línea germinal masculina son factores clave para la espermatogénesis masculina y la fertilidad. Las células sustentaculares (células de Sertoli) como células somáticas juegan un papel fundamental en la creación de un microambiente esencial para la auto-renovación y diferenciación de las células de la línea germinal masculina. Las células madre mesenquimales son reconocidas como células auto-renovables y multipotentes capaces de diferenciarse en múltiples tipos de células. La generación de células germinales masculinas a partir de células madre mesenquimales puede proporcionar un método terapéutico para tratar la infertilidad masculina. En este estudio, las células mesenquimales derivadas de la médula ósea (BMMSCs) se recuperaron de la médula ósea de ratones de 6-8 semanas de edad del Instituto de Investigación Médico Naval (NMRI). En el estudio se aislaron las células sustentaculares y se enrriquecieron usando placas revestidas con lectina. Se obtuvo el medio de condición celular después de diferentes intervalos de tiempo. Posteriormente se cultivaron las BMMSC con diferentes concentraciones de SCCM y medio de Eagle modificado por Dulbecco (DMEM) en diversos momentos. Se evaluaron marcadores específicos de células de línea germinal usando la reacción en cadena de polimerasa transcriptasa inversa (RT-PCR) e inmunocitoquímica. Los resultados mostraron que las BMMSCs cultivadas con SCCM durante 48h exhibieron transcritos específicos de línea germinal (Mvh, Iid4, piwil2) (p <0,05) y marcadores (Mvh, Scp3). Nuestros resultados indican que el cultivo de BMMSCs con SCCM puede conducir a la diferenciación efectiva de BMMSCs en células germinales y proporcionar una estrategia de tratamiento para la infertilidad masculina.

SUMMARY: Male germ line stem cells are key factors for male spermatogenesis and fertility. Sustentacular cells (Sertoli cells) as somatic cells play a pivotal role in creating essential microenvironment for the self-renewal and differentiation of the male germ line cells. Mesenchymal stem cells are recognized as self-renewing and multipotent cells able to differentiate into multiple cell types. The generation of male germ cells from mesenchymal stem cells may provide a therapeutic method to treat male infertility. In this study, Bone marrow derived mesenchymal cells (BMMSCs) were retrieved from the bone marrow of 6-8-week old Naval Medical Research Institute (NMRI) mice. Sustentacular cells (Sertoli cells) were isolated and made rich using lectin coated plates. Sustentacular cell condition medium (SCCM) was collected after different time intervals. Then the BMMSCs were cultured with different concentration of SCCM and Dulbecco's Modified Eagle's medium (DMEM) at various times. Specific markers of Germ line cells were evaluated by using Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. The results showed that BMMSCs cultured with SCCM for 48h exhibited germ line specific transcripts (Mvh, Iid4, piwil2) (p< 0.05) and markers (Mvh, Scp3). Our findings represent that culturing BMMSCs with SCCM may lead to effective differentiation of BMMSCs into germline cells and provide a treatment strategy for male infertility.

Kisspeptin expression features in the arcuate and anteroventral periventricular nuclei of hypothalamus of letrozole-induced polycystic ovarian syndrome in rats.

PURPOSE: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of the reproductive system characterized by polycystic ovaries and androgen excess. Letrozole is a nonsteroidal aromatase inhibitor that is used in experimental research to induce PCOS. Kisspeptin is an essential protein in regulation of cyclicity. Kisspeptin receptor is expressed in the hypothalamus and pituitary glands, and kisspeptin containing neurons are affected from sex steroid hormones. We aimed to investigate the number of kisspeptin-positive cells in the arcuate (Arc) and anteroventral periventricular nuclei (AVPV) of hypothalamus in the letrozole-induced PCOS. METHODS: 40 female Wistar rats were divided into the proestrus control, diestrus control, proestrus vehicle, diestrus vehicle and letrozole. Animals were sacrificed after 3 weeks, and sera, ovary and brain samples were harvested for further evaluations. RESULTS: Letrozole group had high weight gain, high numbers of ovarian follicular cysts, high levels of luteinizing hormone and testosterone and increase number of kisspeptin-positive cells in the Arc nucleus, as compared with the control groups (P ≤ 0.05 vs. proestrus control and proestrus vehicle). Letrozole group showed a decrease in the number of kisspeptin-positive cells in the AVPV nucleus (P ≤ 0.05 vs. proestrus control and proestrus vehicle). CONCLUSION: Our findings show that the number of kisspeptin-positive cells may be affected from letrozole, and that the changes in the number of these cells may be in favor of the appearance of PCOS features in this group.