During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect

During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND) 5th to 15th male rats were daily injected with: 1) 2.5 mg of testosterone propionate (TP), or 2) 12.5 microg of 17beta-estradiol benzoate (EB), or 3) TP+EB, or 4) 7.5 IU of human purified FSH (hFSH), or 5) hFSH+EB or solvents (control-C). Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF) were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7+/-2.0 vs. 30.9+/-1.6 microm2 for C, p<0.001) and increased after hFSH+EB (33.1+/-2.3 microm2, p<0.05). Incidence of LF was completely arrested by steroid hormone treatments given separately, significantly inhibited after TP+EB (median: 0.0%, vs. 2.0% for C p<0.05) and significantly enhanced after hFSH+EB (median: 51.0%, p<0.001). hFSH alone did not influence LF. Incidence of TUNEL positive Sertoli cells significantly increased after EB (median: 2.9% vs. 0.5% for C, p<0.05) or TP+EB (median: 2.2%, p<0.01) and was not affected by other treatments. Incidence of TUNEL positive germ cells increased significantly after EB alone (median: 4.4% vs. 2.5%, for C, p<0.01 ) and was significantly decreased by hFSH+EB (median: 0.5%, p<0.01). CONCLUSIONS: 1) Administration of testosterone or estradiol to immature rats inhibits Sertoli cell maturation. 2) Estradiol stimulates Sertoli and germ cell apoptosis while testosterone has no effect. 3) Testosterone eliminates estradiol--induced germ cell apoptosis when both hormones act in concert. 4) FSH in concert with estradiol, but neither one of the hormone alone, accelerate Sertoli cell differentiation and effectively inhibit germ cell apoptosis. 5) During seminiferous tubule maturation testosterone and the synergistic action of FSH with estradiol support germ cell survival while estradiol alone has an inhibitory, pro-apoptotic effect.     

环磷酰胺对雄性大鼠生殖免疫功能的影响

目的观察环磷酰胺对大鼠睾丸及其细胞免疫的影响,探讨抗肿瘤药物在生殖免疫功能中的机制。方法选用16只15周龄SD大鼠,随机分为对照组和实验组,每组8只;实验组腹腔注射环磷酰胺20mg/kg/d,连续5天,用药两个月后,应用HE染色法研究大鼠睾丸远期组织学变化,用原位缺口末端标记法（TUNEL方法）检测生精小管中生殖细胞凋亡,放射免疫法检测血清睾酮（T）、卵泡刺激素（FSH）、黄体生成素（LH）,流式细胞术进行血液T淋巴细胞亚群分析。结果实验组睾丸生精小管直径缩小、间距增宽、生精上皮变薄、生殖细胞层次和数量减少、生精小管腔多未见精子形成,实验组睾丸生精小管直径、面积、生殖细胞数均显著低于对照组（P〈0.01）;实验组与对照组比较生殖细胞凋亡增多,差异显著（P〈0.01）;实验组与对照组比较血清T明显降低,差异显著（P〈0.01）,血清FSH、LH水平两组间差异无显著性;血液T淋巴细胞亚群分析,实验组与对照组比较CD3＋CD4＋、CD4＋/CD8＋明显降低（P〈0.01）,CD3＋CD8＋明显升高（P〈0.01）。结论环磷酰胺对大鼠睾丸远期损害明显,促进生殖细胞凋亡,降低睾酮的分泌,并抑制T淋巴细胞的免疫功能。     

Effect of growth hormone and induced IGF-I release on germ cell population and apoptosis in the bovine testis

Bovine growth hormone has been used in dairy cattle to increase milk production,but it also increases the twin parturition rate. This effect is mediated by insulin-like growth factor-I (IGF-I), which prevents follicular atresia by hindering apoptosis of granulosa cells. The action of GH and IGF-I on testicular function remains unclear. The goal of this study, therefore, was to verify the effects of short-term administration of GH and induced IGF-I release on the number of testicular germ cells, testicular morphology, and apoptosis in the bovine testis. Twenty Zebu bulls were split into 2 groups. The bulls in Group 1 (n = 10) were treated with 2 subcutaneous injections of bovine GH (500 mg/bull) 7 d apart. Group 2 bulls (n = 10) received placebos under the same protocol. All of the bulls were slaughtered 14 d after the start of treatment. Fragments of the testis were collected, fixed in Bouin's solution, embedded in paraffin, and the sections stained with hematoxilin and eosin. The paraffin-embedded sections were also used for in situ detection of apoptotic cells. Blood samples were collected at slaughter to measure serum levels of IGF-I, FSH and LH. Neither the number of Stage I seminiferous epithelium germ cells and the morphometric parameters (tubular diameter, seminiferous epithelium height, and volumetric proportions of structural components) nor the blood levels of FSH and LH showed a significant difference between the 2 groups. However, the treated animals showed an increase in serum IGF-I (P<0.01). Apoptotic germ cells were detected in the testis of both groups, showing the same pattern and a stage-specific apoptosis pattern. Most of the labeled cells were spermatocytes. The localization of apoptotic germ cells did not differ between groups. These results suggest that short-term administration of GH does not affect bovine spermatogenesis in adult bulls.     

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5?IU/animal; T3 group-100?μg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.     

Cell proliferation and hormonal changes during postnatal development of the testis in the pig

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.     

Effect of vascular endothelial growth factor and testis tissue culture on spermatogenesis in bovine ectopic testis tissue xenografts

Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 microg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.     

Early prepubertal testis criteria, seminiferous epithelium and hormone concentrations as related to testicular development in beef bulls

The present study was conducted to evaluate testis size, spermatogenesis and hormone concentrations before and when peripheral testosterone reached 1 ng/ml as related to further gonad development of beef bulls (n=28). Blood samples were taken weekly starting at 10 weeks (wk) and when testosterone reached 1 ng/ml (AGE1), the left testis was surgically excised. From AGE1 until 54 wk, blood samples were collected to follow basal and GnRH-stimulated hormone profiles. At 54 wk, the second testis was removed. Testosterone reached 1 ng/ml at 20±0.6 wk and, at this developmental state, the seminiferous tubules occupied 57±1.1% of the testis parenchyma. At this phase, 79.3±1.4% of tubule sections had no germ cells and only 2.4±0.3% of the remaining tubules had spermatocytes as the most advanced germ cell type. Also at AGE1, testis size was correlated with the number of Sertoli cells per testis (r=0.67; P<0.05), but not (P>0.05) with the percentage of tubules with germ cells. There was a consistent increase in body weight and testis size throughout the study showing that hemicastration did not impair the development of the bulls. At 54 wk, seminiferous tubules represented 76±0.7% of the testis parenchyma and 72.3±1.7% of tubule sections were found with either round or elongated spermatids. Quantitative criteria of spermatogenesis in the second testis (excised at 54 wk) were not correlated (P>0.05) with the percentage of seminiferous tubules with germ cells in the first testis (excised at AGE1). As determined by regression analysis, testis diameter measured between 30 and 44 wk (AVTD) was associated with AGE1 and testis diameter averaged at 12 wk and AGE1 (R(2)=0.77; P<0.01). Also, AVTD was related to AGE1, testis diameter at 12 wk and concentrations of 17β-estradiol (estradiol; basal+GnRH-stimulated) averaged between 10 wk and AGE1 (R(2)=0.79; P<0.01). Yearling testis weight, in turn, was linked to AGE1 and testis weight at AGE1 (R(2)=0.49, P<0.01). In conclusion, early detection of 1 ng of testosterone/ml, larger testis size and greater estradiol before and at that developmental period positively relate to future testis attributes. When testosterone reached 1 ng/ml, the seminiferous tubules had Sertoli cells, spermatogonia and a few spermatocytes and events occurring before and at that phase are potential markers of testis growth and sperm-producing capacity of sires.     

Effects of prenatal exposure to a 900 MHz electromagnetic field on 60-day-old rat testis and epididymal sperm quality

We investigated the effects of exposure in utero to a 900 megahertz (MHz) electromagnetic field (EMF) on 60-day-old rat testis and epididymis. Pregnant rats were divided into control (CG; no treatment) and EMF (EMFG) groups. The EMFG was exposed to 900 MHz EMF for 1 h each day during days 13 ? 21 of pregnancy. Newborn rats were either newborn CG (NCG) or newborn EMF groups (NEMFG). On postnatal day 60, a testis and epididymis were removed from each animal. Epididymal semen quality, and lipid and DNA oxidation levels, apoptotic index and histopathological damage to the testis were compared. We found a higher apoptotic index, greater DNA oxidation levels and lower sperm motility and vitality in the NEMFG compared to controls. Immature germ cells in the seminiferous tubule lumen, and altered seminiferous tubule epithelium and seminiferous tubule structure also were observed in hematoxylin and eosin stained sections of NEMFG testis. Nuclear changes that indicated apoptosis were identified in TUNEL stained sections and large numbers of apoptotic cells were observed in most of the seminiferous tubule epithelium in the NEMFG. Sixty-day-old rat testes exposed to 900 MHz EMF exhibited altered sperm quality and biochemical characteristics.     

GnRH and / or testosterone induced changes in reproductive activities during nonbreeding season in Calotes versicolor (Daud.)

Administration of Gonadotrophin releasing hormone (GnRH) to male C versicolor during nonbreeding season increases the weight of testis;diameter of testis, seminiferous tubule, Sertoli and Leydig cell nuclei. It also activates the spermatogenic process. Increase in the weight of epididymis and lowered cholesterol level of testis indicate androgen production. Treatment of tesotsterone along with GnRH further enhances the activities of testis as a few spermatozoa appeared in the lumen of seminiferous tubule along with increase in other spermatogenic elements. It may be concluded that the exogenous GnRH can induce reproductive activities during nonbreeding season when the environmental conditions are unfavourable. Testosterone administration has the additive effect on these activities.     

Reversible changes in the testes and epididymides of dog treated with alpha-chlorohydrin.

1. Chronic administration of alpha-chlorohydrin (8 mg/kg body wt for 30 days) caused lesions in the testis of dog. The changes in the germ cells were degenerative. The seminiferous tubule and Leydig cell nuclear diameter were reduced. 2. Epididymal cell height was greatly reduced and the stereocilia had disappeared completely. The lumen was devoid of spermatozoa. 3. Alpha-chlorohydrin administration inhibited the RNA and sialic acid contents in the testes and epididymides of dog. Total cholesterol and lipids/g of testes were increased significantly after alpha-chlorohydrin administration. 4. These effects were reversible. Repopulation of testis tubules occurred following a period of 100 days recovery in dog. Numerous spermatogonia and sperm develop and traverse the epididymides. The RNA, sialic acid, cholesterol and total lipids of testes and epididymides returned to subnormal levels. 5. The possibility of using alpha-chlorohydrin as male contraception is indicated.     

Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

Background  

Cimetidine, refereed as antiandrogenic drug, causes hormonal changes in male patients such as increased testosterone and FSH levels. In the rat testis, structural alterations in the seminiferous tubules have been related to germ cell loss and Sertoli cell death by apoptosis. Regarding the important role of Sertoli cells in the conversion of testosterone into estrogen, via aromatase, the immunoexpression of estrogen receptors-beta (ERbeta) was evaluated in the germ cells of untreated and treated rats with cimetidine. A relationship between ERbeta immunoreactivity and apoptosis was also investigated in the germ cells of damaged tubules.     

Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation

Genes containing the DM domain, a conserved DNA binding motif first found in Doublesex of Drosophila and mab-3 of Caenorhabditis elegans, regulate sexual differentiation in multiple phyla. The DM domain gene Dmrt1 is essential for testicular differentiation in vertebrates. In the mouse, Dmrt1 is expressed in pre-meiotic germ cells and in Sertoli cells, which provide essential support for spermatogenesis. Dmrt1 null mutant mice have severely dysgenic testes in which Sertoli cells and germ cells both fail to differentiate properly after birth. Here we use conditional gene targeting to identify the functions of Dmrt1 in each cell type. We find that Dmrt1 is required in Sertoli cells for their postnatal differentiation, and for germ line maintenance and for meiotic progression. Dmrt1 is required in germ cells for their radial migration to the periphery of the seminiferous tubule where the spermatogenic niche will form, for mitotic reactivation and for survival beyond the first postnatal week. Thus Dmrt1 activity is required autonomously in the Sertoli and germ cell lineages, and Dmrt1 activity in Sertoli cells is also required non-autonomously to maintain the germ line. These results demonstrate that Dmrt1 plays multiple roles in controlling the remodeling and differentiation of the juvenile testis.     

Mice lacking cyclin-dependent kinase inhibitor p19Ink4d show strain-specific effects on male reproduction

p19(Ink4d) is a member of the INK4 family of cyclin-dependent kinase inhibitors, which are important negative regulators of the G1-phase cyclin-dependent kinases CDK4 and CDK6. On a mixed C57BL/6 x 129P2/OlaHsd background, mice deficient for p19(Ink4d) exhibited defects in male reproductive function including testicular atrophy, alteration in serum follicle stimulating hormone, qualitative increase in germ cell apoptosis, and delayed kinetics of meiotic prophase markers (Zindy et al., 2001. Mol Cell Biol 21:3244-3255; Zindy et al., 2000. Mol Cell Biol 20:372-378). In this study, a quantitative assessment of these aspects of reproductive capacity demonstrated relatively mild deficits in p19(Ink4d-/-) males compared to controls. These effects did not dramatically worsen in older males although some seminiferous tubule defects were observed. Following marker-assisted backcrossing into the C57BL/6 background, p19(Ink4d-/-) males did not display defects in testis weights, sperm numbers, serum FSH, germ cell apoptosis, or kinetics of selected meiotic prophase markers. These studies indicate that a reduction in Ink4 family function by the loss of p19(Ink4d) is sufficient to induce mild reproductive defects in male mice with a mixed genetic background, but not in the C57BL/6 genetic background.     

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.     

The effect of gonadotrophins on the 3',5'-AMP levels of seminiferous tubules

The addition of follicle-stimulating hormone (FSH) to isolated tubules from hypophysectomized rats was shown to increase the level of adenosine 3′,5′-monophosphate (3′,5′-AMP). In contrast, luteinizing hormone (LH) exerted no effect in this system. The results presented are consistent with the concept that FSH exerts a direct effect upon cells within the seminiferous tubule, possibly on Sertoli cells, whereas the effects of LH on spermatogenesis are primarily due to the stimulation of androgen production by the interstitial cells of the testis.     

Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice

The four highly homologous members of the C‐terminal EH domain‐containing (EHD) protein family (EHD1‐4) regulate endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4^−/− mice confirmed EHD4 deletion. Ehd4^−/− mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (Day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (Day 31) were seen in the Ehd4^−/− testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium, and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4^−/− males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and EHD4 levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age‐dependent manner. We conclude that high levels of EHD1 in the adult Ehd4^−/− testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of postmitotic and postmeiotic germ cells and implicate EHD protein‐mediated endocytic recycling as an important process in germ cell development and testis function. genesis 48:328–342, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia)

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.     

Developmental and hormonal regulation of the monocarboxylate transporter 2 (MCT2) expression in the mouse germ cells

During spermatogenesis, postmeiotic germ cells utilize lactate produced by Sertoli cells as an energy metabolite. While the hormonal regulation of lactate production in Sertoli cells has been relatively well established, the transport of this energy substrate to the germ cells, particularly via the monocarboxylate transporters (MCTs), as well as the potential endocrine control of such a process remain to be characterized. Here, we report the developmentally and hormonally regulated expression of MCT2 in the testis. At Day 18, MCT2 starts to be expressed in germ cells as detected by Northern blot. The mRNA are translated into protein (40 kDa) in elongating spermatids. Ultrastructural analysis demonstrated that MCT2 protein is localized to the outer face of the cell membrane of spermatid tails. MCT2 mRNA levels are under the control of the endocrine, specifically follicle-stimulating hormone (FSH) and testosterone, and paracrine systems. Indeed, a 35-day-old rat hypophysectomy resulted in an 8-fold increase in testicular MCT2 mRNA levels. Conversely, FSH and LH administration to the hypophysectomized rats reduced MCT2 mRNA levels to the basal levels observed in intact animals. The decrease in MCT2 mRNA levels was confirmed in vitro using isolated seminiferous tubules incubated with FSH or testosterone. FSH or testosterone inhibited in a dose-dependent manner MCT2 mRNA levels with maximal inhibitory doses of 2.2 ng/ml and 55.5 ng/ml for FSH and testosterone, respectively. In addition to the endocrine control, TNFalpha and TGFbeta also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFbeta and TNFalpha, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell-germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.