A1 Spermatogonia Research Articles

Cultivation and transfer of porcine type A spermatogonia

A cell population enriched with type A spermatogonia has been isolated from the boar testes. Cell types occurring during isolation were morphologically characterized, factors maintaining the cultured spermatogonia in the undifferentiated state were studied, and these cells were transferred to sterile recipients preliminarily treated with busulfan. The cells of spermatogenic epithelium cultivated in vitro for 24 h were used for transfer experiments. The transfer efficiency was estimated within 27 and 29 days according to the histological picture of the testes and the isolated cultures. Spermatogenic cells at various developmental stages and a few Sertoli cells and spermatozoa were found on sections and in cell suspensions. Sperm samples could be taken from recipient boars within nine months after the transfer. Microsatellite analysis of DNA showed the endogenous pattern of spermatogenesis. Thus, it was shown that spermatogenic donor cells can restore and maintain spermatogenesis of a recipient for at least 30 days. However, the donor cells were fully forced by the recipient reserve cells, type A0 spermatogonia, within eight to nine months.

Spermatogenic cycle length and spermatogenic efficiency in the gerbil (Meriones unguiculatus).

The gerbil (Meriones unguiculatus) is a rodent native of the arid regions of Mongolia and China. Because the gerbil can be easily bred in laboratory conditions, this species has been largely used as an experimental model in biomedical research. However, there is still little information concerning the testis structure and function in the gerbil. In this regard, we performed a detailed morpho-functional analysis of the gerbil testis and estimated the spermatogenic cycle length utilizing 3H-thymidine as a marker for germ cell progression during their evolution through the spermatogenic process. The stage frequencies of the XII stages characterized according to the acrosome formation and development were (I-XII) 13.8, 10.1, 8.1, 7.8, 4.0, 11.2, 7.5, 7.1, 5.9, 7.6, 8.1, and 8.9. The mean duration of each seminiferous epithelium cycle was determined to be 10.6 +/- 1.0 days and the total duration of spermatogenesis, based on 4.5 cycles, was approximately 47.5 days. The volume density of tubular and interstitial compartments was approximately 92% and 8%, respectively. Based on the volume occupied by seminiferous tubules in the testis and the tubular diameter, about 9 and 18 m of seminiferous tubules were found per testis and per gram of testis, respectively. Twelve primary spermatocytes were formed from each type A1 spermatogonia. The meiotic index was 2.8, indicating that 30% of cell loss occurs during meiosis. The number of Leydig and Sertoli cells per gram of the testis was 28 million and each Sertoli cell was able to support approximately 13 spermatids. The daily sperm production per gram of testis (spermatogenic efficiency) was 33 million. Taken together, these data indicate that, mainly due to the high seminiferous tubule volume density and Sertoli cell support capacity for germ cells, the gerbil presents high spermatogenic efficiency compared with other mammalian species already investigated. The data obtained in the present study might provide the basis for future research involving the reproductive biology in this species.

244.Interaction between bone morphogenetic protein 4 and retinoid signalling in mouse spermatogenesis

Vitamin A (retinol, or ROL) is also essential for normal spermatogenesis in the rat and mouse. Vitamin A-deficient (VAD) rodents suffer various disorders including blindness and male infertility. The molecular mechanisms leading to infertility in vitamin A deficient rodents have never been fully elucidated. Following prolonged vitamin A withdrawal the only germ cells remaining in the VAD rodent testis are stem cell spermatogonia, type A1 spermatogonia, and a few preleptotene spermatocytes. Supplementing the diet of these animals with retinoic acid (RA) alleviates all symptoms of vitamin A deficiency, with the exception of sight and spermatogenesis. It is not until VAD animals are re-administered ROL through the diet, or RA is injected in repeated high doses directly into the testis, that normal spermatogenic function is restored. Here we report an interaction, in germ cells, between the Bone Morphogenetic Protein (BMP) 4 and retinoid signalling pathways that may help explain the molecular mechanics of vitamin A deficiency. We localised BMP4 gene expression to adult germ cells, in particular spermatogonia, at both the mRNA and protein level. We generated VAD mice and found that in the absence of retinoids in vivo, bmp4 gene expression was significantly upregulated in the testis. We also observed that the expression of bmp4 is downregulated by retinoid treatment in germ cells isolated from vitamin A sufficient mice. Expression of bmp4 mRNA in isolated spermatogonia was more sensitive to ROL rather than RA. Our results may reflect a direct requirement for ROL by germ cells for the resumption of spermatogenesis in VAD animals that involves the regulation of BMP4 expression. Furthermore our observations suggest that retinoid signalling in germ cells is different to that observed in somatic cells, and may provide insights into the role of retinoids in spermatogenesis.

Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development.

Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.

Testicular development, histology, and hormone profiles in three yearling angus bulls with spermatogenic arrest

This article discusses the interactions between testis criteria and hormone profiles in Angus bulls with spermatogenic arrest. From 2 to 12 months (mo), testis diameter and hormone concentrations (basal and GnRH-stimulated) were evaluated in 27 bulls. At 12 mo, testes were excised. The z statistical test was used to determine whether parameters in three infertile bulls were different (P < 0.05) from those in 24 bulls with normal spermatogenesis. Bull 1 had Sertoli cell-only syndrome and Bull 2 had 90% of the tubules without germ cells and only A1 spermatogonia in the remaining. In Bull 3, germ cells did not advance beyond the primary spermatocyte stage. At 12 mo, testes of Bull 1 (99 g), Bull 2 (105 g) and Bull 3 (32 g) weighed less than those of normal bulls (251.5 ± 56 g). Sertoli cell numbers/testis in Bull 1 (3.8 × 10 9) and Bull 2 (4.3 × 10 9) were not different from those in normal bulls (4.9 ± 0.3 × 10 9), but were reduced in Bull 3 (1.6 × 10 9). The number of Leydig cells per gram of testis parenchyma was higher in Bull 1 (5.4 × 10 7), Bull 2 (7.3 × 10 7) and Bull 3 (19 × 10 7) than in normal bulls (3.6 ± 0.2 × 10 7). In Bulls 1 and 2, basal and GnRH-stimulated LH, FSH, testosterone (T), androstenedione (Δ 4A) and estradiol 17-β (E 2) were within normal ranges at most ages. However, basal FSH and LH were greater in Bull 3 than in normal bulls, probably the causes for higher Leydig cell density. Also in the same animal, GnRH induced lower responses in LH and FSH, consequence of low basal T and E 2 at some ages. Basal and GnRH-stimulated Δ 4A in Bull 3 were greater than in normal bulls after 6 mo, indicating impairment of Leydig cell differentiation. Deficiency in hormone secretion did not appear to be the cause of infertility, which points toward impaired gonadal responses or secretion of intratesticular factors, or genetic defects. Moreover, infertile animals may not always show pronounced changes in hormone secretion, but evaluation of testis growth around puberty can help identify those animals that do not proper gonadal development.

Proliferation and differentiation of spermatogonial stem cells.

Spermatogonial stem cells (A(s) spermatogonia) are single cells that either renew themselves or produce A(pr) (paired) spermatogonia predestined to differentiate. In turn, the A(pr) divide into chains of A(al) (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A(s), A(pr) and A(al) spermatogonia proliferate during part of the epithelial cycle forming many A(al) spermatogonia. During epithelial stage VIII, almost all A(al) spermatogonia, few A(pr) and very few A(s) spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D(2) and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x(L) are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A(s), A(pr) and A(al) spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.

Proliferation and differentiation of spermatogonial stem cells

Spermatogonial stem cells (A(s) spermatogonia) are single cells that either renew themselves or produce A(pr) (paired) spermatogonia predestined to differentiate. In turn, the A(pr) divide into chains of A(al) (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A(s), A(pr) and A(al) spermatogonia proliferate during part of the epithelial cycle forming many A(al) spermatogonia. During epithelial stage VIII, almost all A(al) spermatogonia, few A(pr) and very few A(s) spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D(2) and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x(L) are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A(s), A(pr) and A(al) spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.

Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia.

The proto-oncogene c-kit is encoded at the white-spotting locus and in the mouse mutations at this locus affect the precursor cells of melanocytes, hematopoietic cells, and germ cells. c-kit is expressed in type A spermatogonia, but whether or not c-kit is present both in undifferentiated and differentiating type A spermatogonia or only in the latter cell type is still a matter of debate. Using the vitamin A-deficient mouse model, we studied messenger RNA (mRNA) and protein expression in undifferentiated and differentiating type A spermatogonia. Furthermore, we quantified the immuno-positive type A spermatogonia in the epithelial stages VI, VII, IX/X, and XII in normal mice to correlate c-kit expression in type A spermatogonia with the differentiation of these cells. Our results show that in the VAD situation undifferentiated type A spermatogonia express little c-kit mRNA. The A spermatogonia with a larger nucleus expressed c-Kit protein, whereas the A spermatogonia with a smaller one did not. After induction of differentiation of these cells into type A1 spermatogonia, c-kit mRNA was enhanced. The percentage of A spermatogonia expressing c-Kit protein did not change during this process, suggesting that A spermatogonia, which are committed to differentiate express c-kit. Under normal circumstances in epithelial stage VI 16%+/-2% (mean +/- SD), in VII 45%+/-15%, in IX/X 78%+/-14% and in XII 90%+/-1.9% of the type A spermatogonia were c-kit positive, suggesting that Aaligned spermatogonia gradually change from c-Kit negative to c-Kit positive cells before their differentiation into A1 spermatogonia. It is concluded that c-kit can be used as a marker for differentiation of undifferentiated into differentiating type A spermatogonia.

Arrest of Spermatogonial Differentiation in jsd/jsd, Sl17H/Sl17H, and Cryptorchid Mice

The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50-70%) was comparable to that seen in epithelial stages VII-VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A(al) spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia. The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger. In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2(n) cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane. It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.

Respostas hormonais ao tratamento com GnRH e estradiol e suas correlações com desenvolvimento testicular e número de células sertoli em touros angus x charolês na fase de pré-puberdade

O objetivo desta pesquisa foi determinar as correlações entre níveis hormonais aos 2 e 6 meses de idade e diâmetro e histologia testicular em tourinhos Angus x Charolês aos 6 meses de idade (n=15). Aos dois meses, duas amostras basais de sangue foram coletadas a intervalos de 1 hora. Após a última coleta, os animais receberam 5 mg de GnRH e 1,5 e 3,0 horas depois novas amostras foram coletadas. Aos seis meses, os tourinhos foram tratados com 1 mg de estradiol benzoato (E) e receberam 10 mg de GnRH doze horas depois. Após cinco dias, os tourinhos receberam 10 mg de GnRH, mas sem pré-tratamento com estradiol. As amostras de sangue foram coletadas 1,5 e 3,0 horas após a injeção de GnRH nas duas ocasiões. Imediatamente antes dos tratamentos com estradiol e GnRH, duas amostras basais foram coletadas. Os animais foram cirurgicamente castrados aos seis meses. Houve correlações entre níveis de FSH estimulados com GnRH aos dois meses e diâmetro testicular e número de células Sertoli e espermatogônias A1 por testículo aos seis meses de idade (r = -0,65 a -0,80). As associações entre FSH aos dois meses e número de seções de túbulos seminíferos sem células germinativas (r = 0,59) e número de túbulos com espermátides circulares (r = -0,55) também foram significativas. As correlações entre níveis de LH e esteróides e parâmetros testiculares não foram significativas. Aos 6 meses, níveis basais de FSH não alteraram 12 horas após estradiol, mas as concentrações de FSH estimuladas com GnRH foram 2,5 vezes mais elevadas, quando se usou estradiol em relação ao uso somente de GnRH. Modelos de regressão com número de células Sertoli, em função dos níveis de FSH após GnRH, mostraram que, quando se utilizaram GnRH + estradiol, obteve-se maior valor de R² (0,43) em comparação à injeção somente de GnRH (0,30). Portanto, concentrações periféricas de FSH são potenciais indicadores do desenvolvimento testicular na fase de pré-puberdade.

The role of the tumor suppressor p53 in spermatogenesis.

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.

Ram lambs need FSH for normal testicular growth, Sertoli cell numbers and onset of spermatogenesis.

The effect of FSH on the development of the testis in the ram lamb was examined in two experiments where lambs were passively immunized against ovine beta-FSH from birth until 100 or 160 d. In both experiments, immunization resulted in a slower testicular growth relative to that of controls. This effect became apparent at around the start of the period of rapid testicular growth (60-70 d of age) and resulted in testicular weights at the end of treatment ranging from 37 to 51% of those of control groups. Within the testis, this was reflected in shorter seminiferous tubules (48-64% of controls) and in lower numbers of Sertoli cells per testis (57-82%). In the rams immunized until 160 d of age, spermatogenesis had begun and immunization against FSH provoked a lower production of germinal cells which was not solely due to the lower number of Sertoli cells but also due to fewer germinal cells being supported by each Sertoli cell. However, the numbers of A0 spermatogonia per testis and the daily production of the A1 spermatogonia were unaffected by immunization, but the production of the B2 spermatogonia and, as a consequence, of leptotene and pachytene spermatocytes and of round spermatids were all markedly lower (43-47% of controls). These effects were not due to any decreases in the secretion of LH or testosterone as seen in the blood levels of these two hormones. These results show that, in the ram lamb, FSH is essential for normal testicular development and for the establishment of a normal population of Sertoli cells. They also confirm that, once spermatogenesis is established, FSH is necessary for a normal production of germinal cells, with one or more of the divisions between the A1 and B2 spermatogonia being sensitive to suppression of FSH.

Mild hypothermia induces apoptosis in rat testis at specific stages of the seminiferous epithelium.

This study was designed to determine the effects of short episodes of mild hypothermia on cell apoptosis in the testis of the adult rat. Apoptosis was assessed by in situ DNA 3'-end labeling in the testes from rats killed 2 hours after cooling (10 degrees C for 30 minutes), quantitated, and compared with spontaneous apoptosis found in control animals. A significantly increased number of dying germ cells appeared after cold treatment at specific stages (stage XIV, P < 0.0001; stage XII, P < 0.001) of the seminiferous epithelium, mainly due to dying primary metaphasic spermatocytes (P < 0.0001), followed by secondary interphase spermatocytes (P < 0.001), and A2 spermatogonia (P < 0.05). The highly specific effect of mild hypothermia on germ cell apoptosis suggests that the process is tightly regulated.

Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes.

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

Characteristics of A spermatogonia and preleptotene spermatocytes in the vitamin A-deficient rat testis.

The proliferative activity and other characteristics of germ cells in the vitamin A-deficient (VAD) rat testis were investigated. In the VAD testis, A spermatogonia and preleptotene spermatocytes were found. The A spermatogonia in the VAD testis showed a bromodeoxyuridine (BrdU) labeling index of 6.6 +/- 1.1% and a mitotic index of 2.8 +/- 0.5%. After continuous labeling with BrdU for up to four days, the ultimate labeling index of A spermatogonia was 11.6 +/- 2.5%, which is less than expected. It is concluded that in the VAD rat testis, many of the proliferating A spermatogonia degenerate. During the first 18 h after administration of vitamin A, no increase was observed in either the labeling index or the mitotic index of the A spermatogonia. However, after 24 h the first wave of A spermatogonia in S phase was found, and the first wave in mitosis was found after 48 h. Furthermore, in the VAD testis the DNA content of most of the A spermatogonia was similar to that of Sertoli cells, i.e., 2n. It is concluded that in the VAD situation, nearly all A spermatogonia are arrested before the S phase of the A1 spermatogonia. The hypothesis is put forward that in the VAD testis, the remaining A spermatogonia are the undifferentiated spermatogonia that are unable to differentiate into A1 spermatogonia. The preleptotene spermatocytes in the VAD testis showed a BrdU labeling index of 20.3 +/- 3.5%, while the DNA content of most of these cells was between 3n and 4n.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of short photoperiodic cycles on male genital tract and testicular parameters in male goats (Capra hircus)

This study was performed in adult male goats in which seasonal variations were abolished by rapid alternations of long days and short days. These treatments have been shown previously to prevent seasonal changes in the hypothalamo-pituitary axis and to maintain testis weight and sperm production at a high level. The experimental groups were exposed for 3 years to an alternation of either a 1 month short (16 h dark; 8 h light) and 1 month long (16 L; 8 D) photoperiod (2 month cycle; n = 5) or of a 2 month short and 2 month long photoperiod (4 month cycle; n = 4). The control groups were maintained in natural photoperiodic conditions (45 degrees N) and goats were slaughtered in the non-breeding season (end of April RS; n = 5) at the same period as light-treated bucks, or in the breeding season (end of September BS; n = 6). The total weight of the testes, the length and mean diameter of the seminiferous tubules of light-treated goats were similar to those in the breeding season, and higher than those in the non-breeding season. The total number of A0 spermatogonia was increased by light treatments as compared to control goats in the breeding and non-breeding season. The daily production of A1 spermatogonia, leptonene primary spermatocytes and round spermatids in light-treated goats was maintained at the peak breeding season level. The intra-testicular concentration of testosterone, total volumes of intertubular tissue and of Leydig cells, and the number of Leydig cells per testis did not differ between groups. Although the mean cross-sectional area of Leydig cells in light-treated goats was similar to this area in non-breeding season goats, it was significantly lower than that of breeding season goats. In conclusion, the rapid alternation of short and long days allowed an increase in all the germ cells from the A0 spermatogonia onwards, which was responsible for the maintenance of high spermatogenetic activity of light-treated goats.

Partial synchronization of spermatogenesis in the immature Djungarian hamster, but not in the immature Wistar rat.

The frequencies of the cellular associations of the seminiferous epithelium were determined at various ages after birth in immature Djungarian hamsters and Wistar rats. The frequencies of the cellular associations present in immature animals were then compared with the frequencies of the corresponding pooled stages in adult animals. At 15 days of age, the cellular associations present in Djungarian hamsters could be divided into four groups based on the presence of A3, intermediate (In), or B (B1 and B2) spermatogonia, or preleptotene-leptotene-zygotene spermatocytes. Compared with adult animals, the group containing spermatocytes was found to be enriched by 52%, while the frequency of the B spermatogonia was reduced by 55%. At 22 days of age, the cellular associations of spermatogenesis were classified into five groups: pachytene spermatocytes associated with A3 spermatogonia. In spermatogonia, B spermatogonia, or preleptotene-leptotene-zygotene spermatocytes, or the meiotic divisions. The stages containing preleptotene-leptotene-zygotene spermatocytes and those containing A3 spermatogonia were enriched by 35% and 59% compared with adult values, respectively. The meiotic divisions were enriched by 21%, but this increase was not significant. The frequency of the B spermatogonia was reduced by 62%. In addition, the synchronization factor, calculated with the computer program SYNTEST, was increased to 1.48 +/- 0.05 (mean +/- SEM) and 1.38 +/- 0.03 at 15 and 22 days of age, respectively, and was significantly higher (P < 0.001 at both ages) than the adult value of 1.08 +/- 0.02. However, after 22 days of age the synchronization factor did not differ from that in adult animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of spermatogenesis in the Indian desert gerbil, Meriones hurrianae (Jerdon): Seminiferous epithelial cycle, frequency of stages, spermatogoniaL renewal and germ cell degeneration

The present study provides information on the germ cell associations, the pattern of spermatogonial cell renewal, percentage frequency distribution of different cellular associations and germ cell degeneration in the adult gerbil, M. hurrianae. Based on the formation of acrosomal system and the development of the spermatids as visualized with PAS-hematoxylin, 15 steps of spermiogenesis were identified. The first 12 steps were associated with 12 stages, and the other three steps were between the first 6 stages. The relative frequency was maximal for stage VII (14.24) and minimal for stage VIII (4.38), indicating stage VII to be of the longest and stage VIII of the shortest duration. On the basis of their shape, size and nuclear morphology, 6 types (A0, A1, A2, A3 In and B) of spermatogonia were identified in the gerbil. A-type spermatogonia are oval in shape. From A1 and A3 spermatogonia, progressive heterochromatinization was evident. The A3-spermatogonia divide to give rise to In spermatogonia which are smaller than A-type cells. The B-type spermatogonia are derived from In-type cells and are round in shape. These divide to give rise to first generation primary spermatocytes. Spermatogonia divide at fixed stages during the CSE, and they exhibit 5 peaks of mitosis. A-type spermatogonia divide during stages VI, IX, XII and I. In- and B-Types divide during stages II and V respectively. During stages XII and I, A3-type cells divide to give rise to In- as well as to A1-type cells, thus restoring the A-spermatogonial population. In this way A3-cells serve to renew the stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal and stage-specific changes in spermatogenesis of rat after gonadotropin deprivation by a potent gonadotropin-releasing hormone antagonist treatment.

GnRH antagonists (GnRH-As) rapidly and reversibly inhibit testicular functions in a variety of experimental models as well as man. Their potential for human male contraception is currently being tested in many centers, including our own. This study was undertaken to provide comprehensive quantitative information on the testes and to document the temporal and stage-specific changes in the kinetics of germ cell degeneration in rats treated daily with the Nal-Glu GnRH-A (1250 micrograms/kg body wt) for up to 4 weeks. Plasma levels of testosterone (T) and the concentrations of testicular T declined to 20.7% and 5.4% of control values, respectively, by 1 week and remained suppressed throughout the treatment period. Preleptotene and pachytene spermatocytes, and step-7 and step-19 spermatids at stage VII were the first germ cells to degenerate soon after (1 week) GnRH-A treatment. Germ cell counts at stage VII also revealed a significant (P < 0.05) reduction in number of preleptotene (25.6%), pachytene (35.4%), and step-7 spermatids (29.1%) in comparison with controls. The number of homogenization-resistant advanced spermatids decreased by 70%. A further progressive loss of spermatogenic activity occurred with time. Treatment with GnRH-A for 4 weeks caused advanced spermatids to decline to nearly undetectable, Step-7 spermatids to decline to 17.7% of the normal level, and the P and PL to decline to 28.6% and 67.7%, respectively, of control values. The number of Sertoli cells and A1 spermatogonia remained unchanged throughout the experimental period. The effects of GnRH-A treatment on spermatogenesis were identical to that of hypophysectomy. These results suggest that: 1) early deprivation of gonadotropins and/or intratesticular T by GnRH-A treatment is followed by stage-specific degeneration of germ cells; 2) pituitary secretions other than LH and FSH have little primary influence on spermatogenesis during early regression; and 3) the GnRH-A-treated rat would be an excellent animal model for studying the targeted effects of LH, FSH, and T on the regulation of spermatogenesis.

Vitamin A-Deficient Testis Germ Cells are Arrested at the End of S Phase of the Cell Cycle: A Molecular Study of the Origin of Synchronous Spermatogenesis in Regenerated Seminiferous Tubules1

Vitamin A deficiency in male rats arrests spermatogenesis and leads to the loss of advanced germ cells. Retinol treatment of these rats seems to result in a synchronous spermatogenesis initiated from the remaining type A1 spermatogonia. To determine at a molecular level the onset of the M phase of the cell cycle occurring in the retinol-treated germ cells, the H1 histone kinase activity associated with the cdc2 kinase/cyclin B complex was measured. This kinase activity is an excellent molecular indicator of mitosis (M phase) since it is known to be high only for approximately 2 h between the G2/M phase transition of the cell cycle and the metaphase of mitosis. This activity was low in vitamin A-deficient testis, increased by 4 h after retinol treatment, and reached a 15.6-fold level at 12 h. These results suggest that the germ cells of vitamin A-deficient testis begin to enter the M phase around 4 h after retinol injection, with the highest percentage of cells entering at 12 h. These results are consistent with placing the origin of synchronous spermatogenesis in regenerated seminiferous tubules at the end of the S phase of the cell cycle.