Immunolocalization of NGF and its Receptors in Ovarian Surface Epithelium of the Wild Ground Squirrel during the Breeding and Nonbreeding Seasons

The ovarian surface epithelium (OSE) plays an important role in normal ovarian physiology. During each reproductive cycle, the OSE takes part in the cyclical ovulatory ruptures and repair. The aim of this study was to investigate the immunolocalization of nerve growth factor (NGF) and its receptors, tyrosine kinase A (TrkA) and p75, in the OSE cells of the wild ground squirrels during the breeding and nonbreeding seasons. There were marked variations in ovarian weight and size between the breeding and the nonbreeding seasons. Histologically, cuboidal cells and squamous cells were identified in the OSE of both seasons. Yet, stronger immunostaining of NGF, TrkA and p75 were observed in cuboidal cells and squamous cells in the breeding season as compared to the nonbreeding season. In addition, plasma gonadotropin concentrations were higher in the breeding season than in the nonbreeding season, suggesting that the expression patterns of NGF, TrkA and p75 in the OSE were correlated with changes in plasma gonadotropins. These findings suggested that NGF and its receptor TrkA and p75 may be involved in the regulation of seasonal changes in the OSE of wild ground squirrel.     

Seasonal Expression of Androgen Receptor,Aromatase, and Estrogen Receptor Alpha and Beta in the Testis of the Wild Ground Squirrel (Citellus Dauricus Brandt)

The aim of this study was to investigate the seasonal expression of androgen receptor (AR), estrogen receptors α and β (ERα and ERβ) and aromatase cytochrome P450 (P450arom) mRNA and protein by real-time PCR and immunohistochemistry in the wild ground squirrel (WGS) testes. Histologically, all types of spermatogenic cells including mature spermatozoa were identified in the breeding season (April), while spermatogonia and primary spermatocytes were observed in the nonbreeding season (June), and spermatogonia, primary spermatocytes and secondary spermatocytes were found in pre-hibernation (September). AR was present in Leydig cells, peritubular myoid cells and Sertoli cells in the breeding season and pre-hibernation with more intense staining in the breeding season, whereas AR was only found in Leydig cells in the nonbreeding season; P450arom was expressed in Leydig cells, Sertoli cells and germ cells during the breeding season, whereas P450arom was found in Leydig cells and Sertoli cells during pre-hibernation, but P450arom was not present in the nonbreeding season; Stronger immunohistochemical signal for ERα was present in Sertoli cells and Leydig cells during the breeding season; ERβ was only expressed in Leydig cells of the breeding season. Consistent with the immunohistochemical results, the mean mRNA level of AR, P450arom, ERα and ERβ were higher in the testes of the breeding season when compared to pre-hibernation and the nonbreeding season. These results suggested that the seasonal changes in spermatogenesis and testicular recrudescence and regression process in WGSs might be correlated with expression levels of AR, P450arom and ERs, and that estrogen and androgen may play an important autocrine/paracrine role to regulate seasonal testicular function.Key words: Wild ground squirrels, testes, seasonal expression, androgen and estrogen receptors, aromatase cytochrome P450, Citellus dauricus Brandt     

Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.     

Increased daily sperm production in the breeding season of stallions is explained by an elevated population of spermatogonia

Seasonal variation in number of spermatogonia and germ cell degeneration was evaluated to determine which mechanism might explain seasonal differences in daily sperm production per testis (DSP/testis) or per g parenchyma (DSP/g) in stallions. Comparing 28 adult stallions (4 to 20 yr old) in each of the nonbreeding (December-January) and breeding (June-July) seasons, the population of type A spermatogonia was more than two times greater (P less than 0.01) in the breeding season. While the number of type B spermatogonia also was elevated (P less than 0.01) in the breeding season, the number of type B spermatogonia/type A spermatogonium was similar (P greater than 0.05) between seasons. Daily sperm production/testis based on each cell type from type B spermatogonia to spermatids with elongated nuclei was lower (P less than 0.01) in the nonbreeding season. Based on DSP/g, there was significant degeneration during the meiotic divisions in the nonbreeding season. However, this reduction in potential spermatozoan production was not significant (P greater than 0.05) when considering DSP/testis. Significant germ cell degeneration also occurred in the breeding season between type B spermatogonia and primary spermatocytes. However, the type A spermatogonial population was sufficiently elevated to override this degeneration and to explain elevated production of sperm in the breeding season of stallions.     

Temporal appearance of seasonal changes in numbers of Sertoli cells, Leydig cells, and germ cells in stallions

The temporal appearance of seasonal changes in numbers of Leydig, Sertoli, and germ cells was evaluated to determine if seasonally increased daily spermatozoan production might be preceded by changes in numbers of either of two somatic testicular cells. A significant increase in numbers of spermatogonia and Sertoli cells preceded the significant increase in number of Leydig cells in the approaching breeding season. Seasonal changes in parenchymal weight and in numbers of Sertoli cells, Leydig cells, and germ cells were maximal in May and June. Numbers of A or B spermatogonia in June were 2.4 to 2.5 times the number present in January. During the same time period, numbers of other germ cells, as well as Leydig cells and Sertoli cells, were increased by 1.5 to 1.9 times. The magnitude of change between January and March (first time period that the change was significant) was greater for A spermatogonia (1.7-fold) than for other cell types (1.3-fold to 1.5-fold). Hence, the need to accommodate more spermatogonial progeny might cause increased testicular size and number of somatic cells, including Sertoli cells. Season did not influence the rate of degeneration between A and B spermatogonia. However, in the breeding season, the conversion of B spermatogonia to primary spermatocytes was reduced. The lack of a seasonal difference in the ratio of primary spermatocytes per Sertoli cell was consistent with a limited capacity of individual Sertoli cells to accommodate primary spermatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal variation in the total volume of Leydig cells in stallions is explained by variation in cell number rather than cell size

Stereological methods were employed in two studies with stallions 1) to determine if seasonal variation in the total volume of Leydig cells is a function of cell number or cell size and 2) to characterize the annual cycle of the Leydig cell population. In the first study, numbers of Leydig cells were calculated for 28 adult (4-20 yr) stallions in the breeding or nonbreeding seasons from nuclear volume density (percentage of the decapsulated testicular volume), parenchymal volume (decapsulated testicular volume), and the volume of individual Leydig cell nuclei. The average volume of the individual Leydig cells was calculated as the total Leydig cell volume/testis (volume density of Leydig cells in the parenchymal volume times parenchymal volume) divided by the number of Leydig cells. The average volume of an individual Leydig cell varied within each season, but means were almost identical for the nonbreeding (6.94 +/- 0.61 picoliter) and breeding (6.91 +/- 0.45 picoliter) seasons. However, Leydig cell numbers per testis were 57% higher in the breeding season, which also had a 58% higher total volume of Leydig cells per testis. In the second study, the numbers of Leydig cells were determined for 43-48 adult horses in each 3-mo period for 12 mo. The number of Leydig cells per testis in May-July was higher (p less than 0.05) than in August-October or February-April, and higher (p less than 0.01) than in November-January. Thus, seasonal fluctuations in the total volume of Leydig cells in adult stallions is a function of the number of Leydig cells that cycle annually.     

Seasonal changes in the expression of PACAP,VPAC1, VPAC2, PAC1 and testicular activity in the testis of the muskrat (Ondatra zibethicus)

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the steroidogenesis and spermatogenesis in the testis through its receptors PAC1, VPAC1 and VPAC2. In this study, we investigated the seasonal expressions of PACAP, PAC1, VPAC1, VPAC2, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and CYP17A1 in the testis of male muskrat during the breeding season and non-breeding season, respectively. Histologically, we observed the presence of Leydig cells, Sertoli cells and various types of germ cells in the testis during the breeding season, yet only Leydig cells, Sertoli cells, spermatogonia and primary spermatocyte during the non-breeding season. In addition, the immunohistochemical localizations of PACAP and VPAC1 were identified in the Leydig cells, spermatogonia and spermatozoa during the breeding season, while only in the Leydig cells and spermatogonia during the non-breeding season, and PAC1 and VPAC2 were localized in the Leydig cells in both seasons, among which LHR, StAR, 3β-HSD and CYP17A1 were also expressed. Meanwhile, the protein and mRNA expression levels of PACAP, PAC1, VPAC1, VPAC2, LHR, FSHR, StAR, 3β-HSD and CYP17A1 in the testis during the breeding season were significantly higher than those during the non-breeding season. These results suggested that PACAP is involved in the regulation of steroidogenesis and spermatogenesis via an endocrine, autocrine or paracrine manner in the testis of muskrat.Key words: Pituitary adenylate cyclase-activating peptide (PACAP), PACAP receptors, steroidogenesis, testis, Ondatra zibethicus.     

Expression of p57 in mouse and human testes

The expression of cyclin-dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.     

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.     

The germ cell development strategy and seasonal changes in spermatogenesis and Leydig cell morphologies of the spiny lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

The viviparous lizards of the Sceloporus genus exhibit both seasonal and continuous spermatogenesis. The viviparous lizard Sceloporus mucronatus from Tecocomulco, Hidalgo, México, exhibits seasonal spermatogenesis. This study demonstrates the relationship between changes in testis volume, spermatogenesis activity, and Leydig cells during the male reproductive cycle of S. mucronatus. A recrudescence period is evident, which starts in the winter when testicular volume is reduced and climaxes in February, when the greatest mitotic activity of spermatogonia occurs. The testicular volume and Leydig cell index increase gradually during the spring with primary spermatocytes being the most abundant cell type observed within the germinal epithelium. In the summer, the secondary spermatocytes and undifferentiated round spermatids are the most abundant germinal cells. The breeding season coincides with spermiogenesis and spermiation; testicular volume also increases significantly as does the Leydig cell index where these cells increase in both cytoplasmic and nuclear volume. During fall, testicular regression begins with a significant decrease in testicular volume and germinal epithelium height, although there are remnant spermatozoa left within the lumen of the seminiferous tubules. During this time, the Leydig cell index is also reduced, and there is a decrease in cellular and nuclear volumes within these interstitial cells. Finally, during quiescence in late fall, there is reduced testicular volume smaller than during regression, and only spermatogonia and Sertoli cells are present within the seminiferous tubules. Leydig cells exhibit a low index number, their cellular and nuclear volumes are reduced, and there is a depletion in lipid inclusion cytoplasmically.     

MTNR1a和MTNR1b基因在繁殖期与非繁殖期雄性高原鼢鼠HPG轴上的表达

高原鼢鼠 (Eospalax baileyi) 终年营地下生活,感光受洞道限制,但褪黑素 (Melatonin) 分泌水平仍存有季节差异,为探明褪黑素对高原鼢鼠季节性繁殖的调控作用,研究利用q?PCR技术检测雄性高原鼢鼠繁殖期 (5月) 和非繁殖期 (9月) 下丘脑、垂体及睾丸中褪黑素受体1a (Melatonin receptor 1a, MTNR1a) 和褪黑素受体1b (Melatonin receptor 1b, MTNR1b) 基因mRNA的相对表达量,通过免疫组织化学技术对MTNR1a和MTNR1b在睾丸中定位,并采用Image Pro Plus软件进行免疫组化阳性评价。结果发现,高原鼢鼠繁殖期下丘脑和垂体中MTNR1a基因的相对表达量显著高于非繁殖期的相对表达量 (P < 0.05),MTNR1b基因的相对表达量在不同时期无显著差异 (P > 0.05),但非繁殖期睾丸中MTNR1a和MTNR1b基因的相对表达量均显著高于繁殖期 (P < 0.01);繁殖期除长形精子外的所有类型细胞以及非繁殖期的间质细胞、支持细胞和精原细胞中均观察到MTNR1a的阳性信号,繁殖期除精原细胞和长形精子细胞外的所有类型细胞,以及非繁殖期间质细胞和支持细胞中均观察到MTNR1b的阳性信号,且非繁殖期MTNR1a和MTNR1b的平均光密度值均显著高于繁殖期 (P < 0.01)。MTNR1a和MTNR1b基因在雄性高原鼢鼠HPG轴上的表达模式,提示了褪黑素在其季节性繁殖调控中的潜在作用。     

Expression of calbindin-D 28k in developing and growing chick testes

Summary Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Seasonal changes in the seminiferous epithelium of rhesus and bonnet monkeys

With a view to elucidate seasonal variations in testicular spermatogenesis, quantitative analysis of spermatogenic cells was carried out in non-human primate species viz. rhesus (Macaca mulatta) and bonnet (M. radiata) monkeys during breeding (October-December) and non-breeding (May-June) seasons. The results revealed significant inhibition of testicular germ cell population during non-breeding compared with the breeding period in both the species. Quantitative determination of Sertoli cell-germ cell ratio showed a marked decrease in the number of type A-spermatogonia, spermatocytes (non-pachytene and pachytene) and spermatids (in steps 1-12 of spermiogenesis) in rhesus monkey during the non-breeding period. Bonnet monkeys exhibited the significant decline in the number of primary spermatocytes and spermatids during the non-breeding phase. In addition, average diameter of round seminiferous tubules and nuclear diameter of Leydig cells also decreased significantly in rhesus monkeys. However, bonnet monkeys did not show any significant change in nuclear diameter/morphology of Leydig cells, testicular tubular diameter and number of type A-spermatogoniae. Sertoli cell number did not show any significant change during both breeding and non-breeding periods in both the species. The results of this study indicate a prominent seasonal variation in testicular spermatogenic/Leydig cells in rhesus monkeys than those observed in bonnet monkeys.     

Expression of calbindin-D28k in developing and growing chick testes.

Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Androgen resistance in squirrel monkeys (Saimiri spp.)

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.     

Localization of inhibin in testes of human, bonnet monkey, dog and rat by immunoperoxidase technique

Immunocytochemical study on the localization of inhibin in the testes of human, bonnet monkey, dog and rat was carried out using indirect immunoperoxidase technique, in order to investigate the cell types involved in inhibin production/storage. A positive reaction was observed in the testes of human, monkey and dog while it was negative in rat testis using specific antiserum to human testicular inhibin generated against homogeneous preparation of human testicular inhibin in our laboratory. Inhibin was found to be localized in Sertoli cells, spermatogonia and primary spermatocytes of human, monkey and dog testes. A weak positive reaction was observed in spermatids of human testis only. Interestingly, Leydig cells of human, monkey and dog testes showed positive reaction indicating presence of inhibin in these cells also.     

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Apoptosis is not the cause of seasonal testicular involution in roe deer

Apoptosis is involved in the regulation of spermatogenesis. The involution of testes in seasonal breeders might be expected to involve enhanced apoptotic cell elimination. We have compared seasonally changing testicular apoptosis in roe deer with that in non-seasonally breeding cattle. Apoptotic cells were detected as TUNEL-positive cells by both flow-cytometric analysis and in situ localisation of fragmented DNA in tissue sections. Apoptosis-induced DNA fragments were also assessed by enzyme-linked immunosorbent assay (ELISA) in homogenised testicular parenchyma. As expected, the testis mass and the percentage of haploid cells in roe deer showed a seasonal pattern with a significant maximum during the rut (August), whereas no annual variation of these parameters was found in bulls. All three methods for determining apoptosis showed similar findings. Roe deer exhibited significant seasonal fluctuation of total apoptotic activity (ELISA, apoptotic cells per tubule cross section) with a maximum during the breeding season. However, the seasonal differences in the number of apoptotic cells corresponded to the variable total numbers of spermatogonia and spermatocytes per tubule cross section. Thus, the percentages of TUNEL-positive cells related to the combined number of both germ cell types showed no seasonal variance, as confirmed by percentages of apoptotic cells analysed flow-cytometrically. The maximum level of apoptosis during the rut in roe deer was similar to the values obtained during the invariably high spermatogenic activity in cattle. These results suggest that, in roe deer, apoptosis is not the cause of the seasonal involution of testes. This study was partially supported by grant Bl 319/6-1 from the Deutsche Forschungsgemeinschaft.