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Biology of Reproduction 66, 14-20 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Follicle-Stimulating Hormone Is Indispensable for the Last Spermatogonial Mitosis Preceding Meiosis Initiation in Newts (Cynops pyrrhogaster)1

Takashi Yazawaa,b, Takashi Yamamoto, Yan Jinb, and Shin-ichi Abéb

a Department of Materials and Life Science, Graduate School of Science and Technology, b Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that mammalian FSH induced differentiation of secondary spermatogonia into primary spermatocytes in organ culture of newt testicular fragments, whereas in medium lacking FSH primary spermatocytes never appeared. Here, we investigated why spermatogonia fail to form primary spermatocytes in the absence of FSH. Spermatogonia maintained proliferative activity and viability at about half the level of those cultured in the presence of FSH, progressed into the seventh generation, but became moribund during the G2/M phase. Thus, the eighth generation of spermatogonia never appeared, suggesting that cell death is the chief reason why primary spermatocytes fail to form in the absence of FSH. The presence of Dmc1, a molecular marker for the spermatocyte stage, confirmed our microscopic observations that spermatogonia differentiated into primary spermatocytes in the presence of FSH. Thus, FSH is indispensable for the completion of the last spermatogonial mitosis, a prerequisite for the conversion of germ cells from mitosis to meiosis. Because prolactin induced apoptosis in spermatogonia during the seventh generation, we propose that a checkpoint exists for the initiation of meiosis in the seventh generation whereby spermatogonia enter meiosis when the concentration ratio of FSH to prolactin is high but fail to do so when the ratio is low.

apoptosis, follicle-stimulating hormone, meiosis, prolactin, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is initiated when spermatogonial stem cells proliferate. After a species-specific number of mitotic divisions, the spermatogonia enter meiosis. Although it is known that this phase is dependent mainly on the action of pituitary gonadotropin and androgens, the regulatory mechanism controlling the initiation of meiosis is poorly understood [1–4].

To address this question, our laboratory has performed a series of studies with the urodelen testis because it offers several advantages. First, its structure is simple compared to anuran and amniote testes. The urodelen testis has several zones, each consisting of a specific spermatogenic stage [1]. Therefore, we can select tissue containing only the spermatogonial stage as starting material for culture. Because the testis is a cystic structure and the number of cells per cyst may be counted, we can determine the number of times spermatogonia have divided in a given cyst. Second, we established in vitro culture systems in which mammalian FSH alone induces the differentiation of secondary spermatogonia into primary spermatocytes in organ [5, 6] and reconstituted [7] cultures in a synthetic medium. We also found that prolactin induces apoptosis in spermatogonia during the seventh mitotic generation [8, 9]. Thus, the newt testis is an excellent model for investigating the roles of FSH and prolactin in spermatogenesis, especially their roles in the initiation of meiosis.

In the current study, we investigated why secondary spermatogonia in testicular fragments never differentiated into spermatocytes when cultured in basal medium lacking FSH. We examined the proliferative activity and viability of spermatogonia at various temperatures, and the number of mitotic divisions they completed in the absence of FSH. We found that spermatogonia maintained proliferative activity and viability into the seventh mitotic generation, but failed to enter the eighth (last) generation preceding meiosis because of cell death. Hence, we suggest that FSH is indispensable for the completion of the last (seventh) spermatogonial mitosis that is prerequisite for the entrance of spermatogonia into meiosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult male newts (Cynops pyrrhogaster), purchased from a dealer (Hamamatsu Seibutsu Kyozai Ltd., Hamamatsu, Japan), were kept at 4°C and transferred to 22°C and incubated for a week until use under 12L:12D illumination and fed frozen Tubifex.

Organ Culture of Testicular Fragments

Newt testes devoid of the anterior tip, which consist of younger stages of spermatogonia, were cut into small pieces (1–2 mm in diameter) and placed on a nucleopore filter (3 pieces/filter; Coaster Corp., Cambridge, MA) in a 35-mm plastic dish (#1008; Falcon, Lincoln Park, NJ). The testicular fragments were cultured with or without porcine FSH (Sigma Chemical Co., St. Louis, MO) at 15, 18, or 22°C in humidified air for 2 wk and fixed in Bouin solution. The basal culture medium consisted of Leibovitz-15 medium supplemented with 10 mM HEPES, adjusted to pH 7.4 with 1 N NaOH.

Histology and Immunohistochemistry

Testes were dehydrated in graded ethanol, embedded in paraffin, and sectioned serially at a thickness of 5 µm. The sections were stained with hematoxylin-eosin. To estimate the number of germ cells (N) in a given spermatocyst, Abercrombie's formula [10] was used: N = n x 5 /(5 + d), where n = the number of the all nuclei in a given cyst on all sections containing the cyst, and d = the average diameter of the largest nuclei in sections.

From our previous study [11], we can estimate the generation of the spermatogonial cyst by counting the number of spermatogonial nuclei in a cyst in a section in which the maximum cyst size is shown as follows; 3–4, 23/cyst (fourth generation); 5–9, 24/cyst (fifth generation); 10–16, 25/cyst (sixth generation); 17–25, 26/cyst (seventh generation); 26–33, 27/cyst (eighth generation).

For immunohistochemistry, sections were incubated with monoclonal anti-5-bromo-2'-deoxyuridine (BrdU; Sigma) or anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. On the following day the sections were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin G (Wako Pure Chemical Industries, Osaka, Japan) for 1 h at room temperature. Color reaction was developed by incubation in 3,3-diaminobenzidine (Sigma) in Tris-HCl buffer (pH 7.4). The sections were counterstained with hematoxylin.

Cell Proliferation Assay

Spermatogonial proliferation was assayed microscopically by immunohistochemical detection of BrdU incorporated into replicating DNA with a kit according to the manufacturer's instructions (Amersham Pharmacia Biotech, Buckinghamshire, UK). Testicular fragments were labeled for 6 h with BrdU and then prepared for histological sections. The proliferative activity of spermatogonia was expressed as the percentage of cysts incorporating BrdU in three sections, each separated by intervals of 24 consecutive sections (around the mid-portion of the fragments). The average ± SEM was obtained for three experiments.

Quantitative Analysis of Survival and Differentiation

To estimate the extent of viability after 2 wk of culture, the number of cysts containing live germ cells was determined for three sections with an interval of 24 sections (around the mid-portion of the fragments) and expressed as a percentage of the 100% that existed in correlative cultures at the beginning of the culture period. An average value ± SEM was obtained for three experiments.

To estimate the extent of differentiation after 2 wk of culture, the cysts containing live germ cells were classified into two groups; cysts consisting of secondary spermatogonia and those consisting of primary spermatocytes. The percentage of spermatocytes reflected the extent of differentiation. An average value ± SEM was obtained for three experiments.

Northern Blot Analysis

Total RNA was prepared from testis fragments by the acid guanidinium thiocyanate-phenol-chloroform method [12]. Total RNA was electrophoresed in a 1% formaldehyde agarose gel and blotted to a nylon membrane, Hybond(N)+ (Amersham Pharmacia Biotech). Antisense digoxigenin-labeled cRNA probe was prepared by in vitro transcription (Boehringer-Mannheim, Mannheim, Germany) of a subclone of newt Dmc1 cDNA. Hybridization was carried out at 68°C overnight in Dig Easy Hyb (Boehringer-Mannheim) containing digoxigenin-labeled cRNA probe, and posthybridization washing was done at 68°C in a buffer of 2x SSC and 0.1% SDS, one more time by transferring the blot into the prewarmed fresh solutions and then twice at 68°C in a buffer of 0.1x SSC and 0.1% SDS. The immunological detection of the signal was carried out according to the manufacturer's instruction (Boehringer-Mannheim).

Statistics

The data were analyzed by the Student t-test. A probability level of <0.05 indicated a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Proliferation and Viability of Secondary Spermatogonia in Basal Medium

In urodeles, spermatogenesis occurs within a cyst consisting of germ cells and Sertoli cells [1]. The germ cells in each cyst are considered to be a clonal population derived from a single primary spermatogonium [13, 14], and all spermatogonia within a cyst incorporate BrdU synchronously when they synthesize DNA [15]. After newts were incubated at 22°C for a week following transfer from 4°C, the testis fragments used for culture consisted of the fourth (23/cyst) through sixth (25/cyst) generations of spermatogonia.

First we investigated whether secondary spermatogonia cultured in basal medium lacking FSH maintain proliferative activity at various temperatures (15, 18, and 22°C; Fig. 1). After 1 wk the proliferative activity decreased significantly at all temperatures examined, but still maintained about half its initial value (S) and the value in FSH-supplemented medium. In the presence or absence of FSH, the highest labeling index was observed at 18°C, and was significantly higher than that at 22°C, but there were no significant differences between 15°C and other temperatures.



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  		FIG. 1. Proliferative activity of secondary spermatogonia after 1 wk of testicular organ culture with (F) or without (C) FSH at various temperatures. Start (S), on Day 0 of culture. Vertical bars represent standard errors of the mean in the three experiments. Values with the same letters do not differ significantly from each other at the 5% level

Next, we examined whether spermatogonial viability is maintained after 2 wk of culture lacking FSH. The viability of cysts remarkably decreased without FSH at all temperatures examined, but was higher at lower temperatures (Fig. 2). In the presence of FSH, the viability was highest at 18°C and lowest at 22°C.



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  		FIG. 2. Viability of the germ cells in testes cultured with (F) or without (C) FSH for 2 wk. Start (S), on Day 0 of culture. White column, Cysts consisting of secondary spermatogonia; shaded column, cysts consisting of primary spermatocytes. Values with the same letters do not differ significantly from each other at the 5% level

Spermatogonia Proceeded to the Seventh Generation but Never Differentiated into Primary Spermatocytes in Basal Medium

The testis fragments used for culture initially consisted of the fourth (about 14%), fifth (about 60%), and sixth (about 26%) generations of spermatogonia in three experiments. So, the most advanced stage was the sixth generation (Fig. 3A).



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  		FIG. 3. Histogram showing number (percentage) of the cysts comprising 23, 24, 25, 26, and 27 germ cells/cyst and photomicrographs of the cells before (A) and after culture with (B) and without (C) FSH for 2 wk at 18°C. The total number of the cysts counted were 126 in A, 109 in B, and 102 in C. Arrowheads in C show the dead nuclei of spermatogonia. Panel D Shows the precise number of cysts in the most advanced stage in basal medium. Bars = 50 µm

In the presence of FSH, primary spermatocytes did differentiate when cultured for 2 wk at 18°C and 22°C (Figs. 2 and 3B). The fragments contained the sixth (25/cyst; about 8%) and seventh (26/cyst, about 30%) generations of spermatogonia and primary spermatocytes (27/cyst; about 62%; Fig. 3B). These morphological observations were confirmed by detection of a 2-kilobase transcript of Dmc1 mRNA (Fig. 4), a molecular marker for spermatocytes [11]. The signal was very faint at 1 wk, but became much stronger by 2 wk. In contrast, in the absence of FSH, primary spermatocytes never formed even by 2 wk (Fig. 3C), although the proliferation and viability of spermatogonia were maintained to some extent at all temperatures examined (Figs. 1 and 2). No signal of Dmc1 mRNA was detected in testis fragments before or after culture in the basal medium (Fig. 4). Then we determined the number of spermatogonial mitoses that occurred at 2 wk of culture without FSH. The testis fragments consisted of the fifth (about 4%), sixth (about 32%), and seventh (about 64%) generations of spermatogonia (Fig. 3C). Precise counting of the number of spermatogonial nuclei in a cyst showed that the most advanced stage was the seventh generation (Fig. 3D).



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  		FIG. 4. Expression of Dmc1 mRNA in testes cultured for 2 wk in the absence and presence of FSH. Five micrograms of total RNA were electrophoresed in each lane, subjected to blotting, and hybridized with Dig-labeled newt Dmc1 cDNA (upper panel). The asterisk (*) indicates the positions of 28S (upper panel) and 18S (lower panel) rRNA. Lower panel shows the ethidium bromide-stained pattern of rRNA that verifies approximately equal loading of total RNA

Apoptosis Occurs in the Seventh Generation of Spermatogonia in Basal Medium

Apoptotic cells with condensed chromatin [8, 9] were often observed in fragments cultured in basal medium (Fig. 5A). It was difficult to count the number of apoptotic nuclei in a cyst correctly, because some nuclei may have been phagocytosed by Sertoli cells. The flanking and other live cysts in the same lobule comprised the seventh generation of spermatogonia (Fig. 5A), indicating that the apoptotic cysts consisted of the seventh generation of spermatogonia. In such lobules, PCNA [16] staining in the nuclei of living germ cells showed a strong or spotty pattern characteristic of the S/G2 phase of the cell cycle (Fig. 5B) [11]. Finally, cysts consisting of moribund nuclei and those containing mitotic figures were in the same lobule (Fig. 5C). These results show that although secondary spermatogonia cultured without FSH proliferate to reach the seventh generation, and progressed to near the G2/M phase, they never completed the last division, but instead died. Consequently, primary spermatocytes never formed.



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  		FIG. 5. Photomicrographs of spermatogonia in the seventh generation in sections of testes cultured for 2 wk at 18°C in basal medium. A) In a lobule, cysts comprising apoptotic nuclei of spermatogonia (circled by solid lines) are flanked with those in the seventh generation of living spermatogonia (circled by dotted lines); B) immunostaining obtained with anti-PCNA antibodies in the nuclei of living spermatogonia showed a strong or spotty pattern characteristic of the S/G2 phase of the cell cycle; C) in a lobule, a cyst containing mitotic figures is flanked with that consisting of moribund cells. Bars = 50 µm (A, B), 10 µm (C)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we clarified why newt spermatogonia do not differentiate into primary spermatocytes in cultures lacking FSH. When FSH was absent, spermatogonia displayed decreased proliferation, yet proceeded into the seventh mitotic division, but failed to complete mitosis and died. Thus, we identified a specific time when FSH acts and showed that it is indispensable for the last spermatogonial division preceding the initiation of meiosis, while it also promotes proliferative activity and viability of spermatogonia. The death of spermatogonia then prevents further differentiation and no primary spermatocytes form.

Heterologous mammalian FSH was used in the current study because newt FSH was not available. However, we have previously demonstrated that mammalian FSH stimulates the proliferation of newt secondary spermatogonia and their differentiation into primary spermatocytes [5, 6]. Recently, Nakayama et al. [17] cloned newt FSH receptor cDNA, and showed that mammalian cells, transiently transfected with the cDNA, displayed specific binding to mammalian FSH and cAMP accumulation. In addition, as well as in mammalian testis, newt FSH receptor is expressed on the Sertoli cells [17]. These several lines of evidence strongly indicate that mammalian FSH mimics the action of the newt's own FSH.

Several studies have suggested that FSH is crucially important for the regulation of spermatogenesis in adult primates [18–21], seasonal breeders [22, 23], and juvenile rodents [24–28]. The studies in adult rhesus monkeys [18, 19, 21] indicate that FSH specifically amplifies the number of differentiated spermatogonia. In photo-inhibited Djungarian hamsters, FSH qualitatively restored normal spermatogenesis despite low intratesticular testosterone [23], or induced testicular growth comparable to that in animals transferred to a stimulatory photo period [22]. In contrast, in adult rats, there are conflicting reports on the role of FSH: Immunoneutralization of endogenous FSH causes little effect on spermatogenesis [29, 30] and addition of testosterone alone maintained or restored the spermatogenesis of adult rats in which both LH and FSH levels are markedly suppressed [31, 32]. However, it was shown that spermatogenesis is not quantitatively restored or maintained by exogenous administration of high doses of testosterone in GnRH-immunized and hypophysectomized rats [33–36], and FSH replacement corrected germ cell number, increased the number of B spermatogonia available for entry into meiosis, and maintained the number of preleptotene spermatocytes throughout the treatment [27]. The studies on knockout mouse for the FSHß subunit [37] and FSH receptor [38, 39] genes have demonstrated that FSH signaling is required for maintaining normal testicular size, seminiferous tubular diameter, sperm number, and motility, although the mutant males are fertile. The reasons for the fertility may be a species difference or compensation by other hormones. In vitro studies showed that FSH stimulates mouse [40] and immature rat [26] spermatogonia. Also in the adult rat, FSH was suggested to have a regulatory role in apoptosis and DNA synthesis in a stage-specific manner [41]. However, whether FSH is indispensable for entry into meiosis was not known until our current studies revealed this, at least in newts.

The cell death of newt spermatogonia that we observed probably clarifies some previous findings. Newt spermatocytes disappeared after hypophysectomy and spermatocytogenesis ceased during the cool season [42, 43]. In fact, in this latter study, degenerating cells appeared first in lobules at a stage between secondary spermatogonia and leptotene spermatocytes after hypophysectomy. The reason for this should be absence of FSH, as clarified in our current study. In dogfish, spermatogonia degenerated after surgical removal of the ventral lobe of the pituitary [44–46]. In the rat, immunoneutralization of FSH led to apoptotic cell death in spermatogonia and pachytene spermatocytes, although the time at which the spermatogonia died was not revealed [47, 48]. In knockout mice for the FSH receptor, germ cells in the 2C compartment may have been undergoing increased apoptosis [39]. Thus, there may exist among vertebrates a species-specific spermatogonial stage that is dependent for survival on pituitary FSH.

Spermatogonial death in newts was also induced by prolactin both in vivo [8, 49–52] and in vitro [8, 9], and at a specific time, in the seventh generation, the same stage at which spermatogonia die in basal medium. In newts maintained at low temperature the prolactin concentration in the blood was elevated, which in turn caused apoptosis of spermatogonia [52]. Taken together, our current results suggest that there is a regulatory checkpoint for spermatogonia to differentiate into spermatocytes during the seventh generation of spermatogonia (Fig. 6). When the FSH:prolactin concentration ratio is high (in vitro or warm season), spermatogonia proceed through seven mitotic divisions to enter meiosis, but when the ratio is low (in vitro or cool season) spermatogonia cannot complete the last mitosis and instead die by apoptosis. Because newt FSH receptor is also present on Sertoli cells [17], this checkpoint seems to be regulated by Sertoli cells, although prolactin receptor is expressed by both Sertoli cells and germ cells (unpublished data); which cell type has a functional prolactin receptor is currently unknown. It is conceivable that one or more survival factors and/or differentiation factors produced by Sertoli cells upon stimulation by FSH may circumvent apoptosis in spermatogonia and promote them to complete the last mitosis so that they can enter meiosis. Cessation of spermatocytogenesis by cell death also occurs in shark [53] and frog [54, 55] testes in the spring months. So, we assume that this regulatory checkpoint is conserved in evolution, at least from chondrochytes to amphibians, and contributes to seasonal control of spermatogenesis in poikilothermic vertebrates. Whether this regulatory checkpoint is retained in mammals is unknown.



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  		FIG. 6. Hypothesis on a checkpoint for entry into meiosis. When the FSH:prolactin concentration ratio is high, spermatogonia proceed through the seventh division to enter meiosis, whereas they fail to do so and instead go to die when the ratio is low


   ACKNOWLEDGMENTS
 
We thank Prof. Marie A. DiBerardino for critically reading and editing the manuscript. We also thank Dr. S. Izuta, Kumamoto University, for providing anti-PCNA antibody.


   FOOTNOTES
 
First decision: 2 July 2001.

1 This work was supported by a Grant-in-Aid for Science Research (12680718) from the Ministry of Education, Science, Sports, and Culture of Japan; and by Special Coordination Funds for Promoting Science and Technology. Back

2 Correspondence: Shin-ichi Abé, Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, Kurokami 2-39-1, Kumamoto 860-8555, Japan. FAX: 81 96 342 3437; abeshin{at}gpo.kumamoto-u.ac.jp Back

Accepted: August 9, 2001.

Received: June 12, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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