α‐6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α‐6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self‐renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two‐step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α‐6 integrin by flow cytometry and real‐time RT‐PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two‐step enzymatic digestion. An average of 1 × 10⁵ viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α‐6 integrin expression. Flow cytometry analysis demonstrated no differences in the α‐6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real‐time PCR analysis (p > 0.05). In addition to α‐6 integrin, the expression of GFRa‐1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α‐6 integrin expression.     

Characteristics of testicular cell development of 5‐day‐old mice in culture in vitro

The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5‐day‐old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5‐day‐old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO₂ atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli‐like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast‐like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia‐like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.     

Persistence of undifferentiated spermatogonia in aged Japanese Black cattle

Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age-related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age-related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

羊驼睾丸细胞凋亡及凋亡相关蛋白的定位

本研究旨在观察羊驼睾丸的出生后发育和精子发生过程中的细胞凋亡及凋亡相关蛋白Bcl2和Caspase3 的定位.取材新生、12月龄和24月龄羊驼的睾丸,用TUNEL法检测睾丸发育和精子发生过程的细胞凋亡,用免疫组织化学技术检测凋亡相关蛋白Bcl2和Caspase3在羊驼出生后发育和精子发生过程中的定位.结果显示在新生羊驼睾丸未检测到TUNEL阳性细胞,Caspase3和Bcl2表达于间质细胞,提示在新生期凋亡蛋白参与间质细胞凋亡的调节,为曲精小管的发育提供空间;12月龄羊驼睾丸TUNEL阳性细胞定位于曲精小管中央部分,Caspase3 和Bcl2定位于间质细胞和曲精小管中央生殖细胞,提示在青春期(12月龄)羊驼睾丸,细胞凋亡和凋亡相关蛋白参与曲精小管管腔形成的调节;24月龄羊驼睾丸TUNEL阳性细胞定位于精原细胞、精母细胞和精子细胞,Caspase3 和Bcl2定位于间质细胞和各个发育阶段的生精细胞,Caspase3阳性细胞在精原细胞最高,向精母细胞和精子细胞逐渐减少,Bcl2在精原细胞弱阳性表达,在血睾屏障以内的曲精小管近腔室部分呈弥散性强阳性表达,提示在性成熟(24月龄)羊驼睾丸精子发生过程中,细胞凋亡主要发生于精原细胞和早期精母细胞,Bcl2可能抑制精母细胞之后生殖细胞的凋亡.结果提示在羊驼睾丸出生后发育和精子发生过程中存在细胞凋亡现象;凋亡蛋白Caspase3和Bcl2参与羊驼睾丸发育和精子发生过程中细胞凋亡的调节.     

Immunohistochemical analysis of cyclins in canine normal testes and testicular tumors

The expression of cyclins A, D1, D2 and E were examined immunohistochemically in 5 canine normal testes and 31 testicular tumors, including 14 seminomas, 11 Sertoli cell tumors and 6 Leydig cell tumors. In canine normal testes, cyclin A expression was detected in spermatogonia and primary spermatocytes. This suggests that A-type cyclins may play some role in canine spermatogenesis. Cyclin A expression was also observed in 13/14 (92.9%) seminomas and 2/11 (18.2%) Sertoli cell tumors, but no positive reaction was observed in Leydig cell tumors. Parallel examinations for cyclins D1, D2 and E gave negative results in canine normal testes and testicular tumors. High levels of cyclin A expression in canine seminomas indicate that the neoplastic germ cells may be arrested at the spermatogonia and primary spermatocyte stages of differentiation.     

Expression of retinoic acid‐metabolizing enzymes,ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1 in canine testis during post‐natal development

Mammalian spermatogenesis involves highly regulated temporal and spatial dynamics, carefully controlled by several signalling processes. Retinoic acid (RA) signalling could have a critical role in spermatogenesis by promoting spermatogonia differentiation, adhesion of germ cells to Sertoli cells, and release of mature spermatids. An optimal testicular RA concentration is maintained by retinaldehyde dehydrogenases (ALDHs), which oxidize RA precursors to produce RA, whereas the CYP26 class of enzymes catabolizes (oxidize) RA into inactive metabolites. The objective was to elucidate gene expression of these RA‐metabolizing enzymes (ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1) and their protein presence in testes of young, peripubertal and adult dogs. Genes encoding RA‐synthesizing isozymes ALDH1A1, ALDH1A2 and ALDH1A3 and RA‐catabolizing isomers CYP26A1, CYP26B1 and CYP26C1 were expressed in testis at varying levels during testicular development from birth to adulthood in dogs. Based on detailed analyses of mRNA expression patterns, ALDH1A2 was regarded as a primary RA‐synthesizing enzyme and CYP26B1 as a critical RA‐hydrolysing enzyme; presumably, these genes have vital roles in maintaining RA homeostasis, which is imperative to spermatogenesis and other testicular functions in post‐natal canine testis.     

Characterization and in vitro culture of male germ cells from developing bovine testis

The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.     

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini‐pig

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini‐pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line‐derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha‐1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo‐derived thioguanine‐ and ouabain‐resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT‐PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1‐, DBA‐ and CDH1‐positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.     

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.     

小鼠实验性隐睾诱发生殖细胞类型变化

利用 3 0～ 3 5日龄昆白系小鼠制作实验性隐睾 ,定期分批朴杀取样 ,检查隐睾组织学及生殖细胞群体变化 ,为生殖细胞富集及提高体内精原干细胞转基因效率提供条件和依据。结果表明 ,盆腔隐睾精子发生被阻断于精子形成阶段 ;经历 1 5d以上 ,曲细精管内精子数量较少 ;腹腔隐睾精子发生被阻断于精原细胞向精母细胞过渡阶段 ;经历 3 0 d以上 ,曲细精管仅由精原细胞、少量精母细胞及支持细胞组成。由此可知 ,制作盆腔隐睾 ,可得到含少量精子的生殖细胞群体以及主要含精原细胞的生殖细胞群体     

Enhanced Proliferation of Undifferentiated Spermatogonia after Treatment of Short Photoperiod Exposure in the Syrian Hamster, Mesocricetus auratus

Five Syrian hamsters were exposed to a short photoperiod (8L:16D) during 159 days. Atrophied testes were removed, fixed in Allen's solution; paraffin sections of the testicular tissues and whole-mounted seminiferous tubules were prepared. The numbers of various types of spermatogonia were investigated and compared with those in animals maintained in natural photoperiod (12L:12D). All the types of differentiated spermatogonia (A1, A2, In, B1, B2) were significantly decreased in number after the treatment of short photoperiod exposure, while undifferentiated spermatogonia (isolated, paired and aligned type) were significantly increased at stages V-VI and VII-VIII of the seminiferous epithelial cycle. This strictly local reaction of the undifferentiated spermatogonia to the loss of the differentiated spermatogonia suggests the presence of a feedback effect of a certain type(s) of differentiated cells to the undifferentiated spermatogonial proliferation. This feedback mechanism may also play an important role for regulating annual changes in spermatogenesis of seasonal breeders, not only in laboratory but also in natural habitat.     

Transduction and Transplantation of Spermatogonia into the Testis of Ram Lambs through the Extra-testicular Rete

Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.     

Co‐expression of PACAP with VIP,SP and CGRP in the Porcine Nodose Ganglion Sensory Neurons

Our previous study revealed the expression of substance P (SP) and calcitonin gene‐related peptide (CGRP) in sensory distal ganglion of the vagus (nodose ganglion) neurons in the pig. As these neuropeptides may be involved in nociception, the goal of these investigations was to determine possible expression of vasoactive intestinal polypeptide (VIP), SP and CGRP in the pituitary adenylate cyclase‐activating polypeptide‐immunoreactive (PACAP‐IR) porcine nodose perikarya. Co‐expression of these substances was examined using a double‐labelling immunofluorescence technique. To reveal the ganglionic cell bodies, the pan‐neuronal marker protein gene product 9.5 (PGP 9.5) was used. Quantitative analysis of the neurons revealed that 67.25% of the PGP 9.5+ somata in the right‐side ganglion and 66.5% in the left side, respectively, co‐expressed PACAP‐IR. Moreover, 60.6% of the PACAP‐IR cells in the right‐side ganglion and 62.1% in the left, respectively, co‐expressed VIP. SP‐IR was observed in 52.2 and 39.9% of the right and left ganglia, respectively. CGRP was found in 27.7 and 34.1% of the right and left distal ganglion of the vagus, respectively. High level of co‐expression of PACAP with VIP, SP and CGRP in the distal ganglia of the vagus sensory perikarya directly implicates studied peptides in their functional interaction during nociceptive vagal transduction.