Antagonistic regulation of spermatogonial differentiation in zebrafish (Danio rerio) by Igf3 and Amh.

Fsh-mediated regulation of zebrafish spermatogenesis includes modulating the expression of testicular growth factors. Here, we study if and how two Sertoli cell-derived Fsh-responsive growth factors, anti-Müllerian hormone (Amh; inhibiting steroidogenesis and germ cell differentiation) and insulin-like growth factor 3 (Igf3; stimulating germ cell differentiation), cooperate in regulating spermatogonial development. In dose response and time course experiments with primary testis tissue cultures, Fsh up-regulated igf3 transcript levels and down-regulated amh transcript levels; igf3 transcript levels were more rapidly up-regulated and responded to lower Fsh concentrations than were required to decrease amh mRNA levels. Quantification of immunoreactive Amh and Igf3 on testis sections showed that Fsh increased slightly Igf3 staining but decreased clearly Amh staining. Studying the direct interaction of the two growth factors showed that Amh compromised Igf3-stimulated proliferation of type A (both undifferentiated [Aund] and differentiating [Adiff]) spermatogonia. Also the proliferation of those Sertoli cells associated with Aund spermatogonia was reduced by Amh. To gain more insight into how Amh inhibits germ cell development, we examined Amh-induced changes in testicular gene expression by RNA sequencing. The majority (69%) of the differentially expressed genes was down-regulated by Amh, including several stimulators of spermatogenesis, such as igf3 and steroidogenesis-related genes. At the same time, Amh increased the expression of inhibitory signals, such as inha and id3, or facilitated prostaglandin E2 (PGE2) signaling. Evaluating one of the potentially inhibitory signals, we indeed found in tissue culture experiments that PGE2 promoted the accumulation of Aund at the expense of Adiff and B spermatogonia. Our data suggest that an important aspect of Fsh bioactivity in stimulating spermatogenesis is implemented by restricting the different inhibitory effects of Amh and by counterbalancing them with stimulatory signals, such as Igf3.

Androgen receptor signalling in peritubular myoid cells is essential for normal differentiation and function of adult Leydig cells.

Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM-ARKO) did not affect LC number, but resulted in compensated LC failure. The current study extends these investigations, demonstrating that PTM AR signalling is important for normal development, ultrastructure and function of adult LCs. Notably, mRNAs for LC markers [e.g. steroidogenic factor 1 (Nr5a1), insulin-like growth factor (Igf-1) and insulin-like factor 3 (Insl3)] were significantly reduced in adult PTM-ARKOs, but not all LCs were similarly affected. Two LC sub-populations were identified, one apparently 'normal' sub-population that expressed adult LC markers and steroidogenic enzymes as in controls, and another 'abnormal' sub-population that had arrested development and only weakly expressed INSL3, luteinizing hormone receptor, and several steroidogenic enzymes. Furthermore, unlike 'normal' LCs in PTM-ARKOs, the 'abnormal' LCs did not involute as expected in response to exogenous testosterone. Differential function of these LC sub-populations is likely to mean that the 'normal' LCs work harder to compensate for the 'abnormal' LCs to maintain normal serum testosterone. These findings reveal new paracrine mechanisms underlying adult LC development, which can be further investigated using PTM-ARKOs.

Spermatogonial morphology, kinetics and niches in hamsters exposed to short- and long-photoperiod.

Previous studies have shown that under short photoperiod exposure spermatogenesis in golden hamster regresses leading to sexual inactivity. It is known that this regression is related to changes in somatic and germ cells (spermatocytes and spermatids). However, the photoperiod effects on spermatogonial biology have not been studied in detail yet. In this regard, this study was carried out to investigate the morphology, kinetics and niches of different spermatogonial types in golden hamsters under long- and short-photoperiod. Six spermatogonial generations such as type A undifferentiated (A(und)), type A differentiating (A(1), A(2), A(3)), intermediate (In) and type B spermatogonia were characterized, and were morphologically similar irrespective of the photoperiod exposure. The short photoperiod was inhibitory to A(und) spermatogonia and preleptotene but had no effect on the number of differentiating (A(1) to B) spermatogonia. In golden hamsters exposed to stimulatory-photoperiod, the interstitial components were positioned mainly in triangular areas around the seminiferous tubules and, in this situation, the A(und) spermatogonia were clearly positioned in niches (p < 0.05) in all stages studied. On the other hand, during the inhibitory-photoperiod where the seminiferous tubules have smaller diameter, the interstitial components were more homogenously distributed and the triangular areas were not clearly observed. In this case, the niches were identified only at stage VII (p < 0.05), although there was a trend of being positioned in niches area in all the stages studied. Thus, these findings suggest that the A(und) spermatogonia location in the seminiferous epithelium and the niche position are directly related to the position of the interstitial components.

Neonatal hypothyroidism causes delayed Sertoli cell maturation in rats treated with propylthiouracil: evidence that the Sertoli cell controls testis growth.

BACKGROUND: The testes of rats treated neonatally with propylthiouracil (PTU) grow to almost twice their normal size. The cause of testicular enlargement has been suggested to be the result of delayed maturation of Sertoli cells, allowing Sertoli cell division to occur beyond the 15th postnatal day, the commonly recognized cutoff date for Sertoli cell divisions. It has been shown that an increased population of Sertoli cells in postnatal development supports increased numbers of germ cells in adult animals. After examining developing rats treated neonatally with PTU, we hypothesized that an approximate 10-day delay in maturation was occurring and proceeded to test this hypothesis experimentally. Thus the purpose of this report was to determine if a 10-day delay in maturation could explain the increased numbers of Sertoli cells and increased testis size in PTU-treated animals. METHODS: Both control animals and animals treated neonatally with PTU N = 5/group were sacrificed at 15 and 25 days of age and prepared for electron microscopy. RESULTS: Micrographs show and morphometric ultrastructural analysis of numerous parameters demonstrated at the 95% probability level that Sertoli cells from 25-day-old PTU animals are not different in size and most constituents (volume and surface area) from 15-day-old control animals and are less mature than 25-day-old control animals. Mitosis of Sertoli cells was observed in PTU-treated animals in 25-day-old animals but not in age-matched controls. The number of Sertoli cells in 25-day-old PTU-treated animals is significantly increased over age-matched controls. Micrographs show the presence of immature Sertoli cell nuclei in 25-day-old animals receiving PTU as well as increased germ cell degeneration in this group. Sertoli cell tight junction formation is also delayed in PTU-treated animals as compared with controls. CONCLUSIONS: Together, the data show that delayed maturation of Sertoli cells occurs in treated animals that corresponds to a minimum of 10 developmental days. In the immature state, Sertoli cells continue to divide. Data presented herein and published data related to PTU treatment indicate that delayed maturation of the Sertoli cell results in delayed maturation and proliferation of other testicular cell types. From this and from published data, the hypothesis is presented that the Sertoli cell is responsible for the overall control of testis development.

Building a testis.

Specific cellular, subcellular and acellular components of the rat testis including the capsule, the peritubular tissue (tunica propria) and the lymphatic endothelium were analyzed using morphometric techniques at cellular and subcellular levels to yield volume and surface area data. These data were integrated with previously published data for other cellular components of the rat testis to provide information about the volumetric composition for virtually every component of this organ. For major cell types (Leydig, Sertoli, myoid cells and germ cells) the data are expressed to the subcellular level in terms of volume and, in some instances, surface area. Graphic portrayals of testis constituents are used for rapid visual understanding of testis structure. The data presented herein are useful in conjunction with biochemical data to describe physiological properties of cells and cell components and also for understanding how structure differs under experimental and in pathological situations.

Characteristics of mitotic cells in developing and adult testes with observations on cell lineages.

This report describes characteristics of dividing cells, primarily in developing (10-40 day) rat testis and relates the structure of the dividing cells to the structure of interphase cells. Mitotic cells were characterized in seven zones. Dividing Sertoli cells were seen prior to day 15 and possessed distinct characteristics as compared with dividing germ cells. Myoid cells showed morphological characteristics of precursor myoid cells; 'clear cells' self-replicated in the myoid cell layer; adult-type Leydig cells, some containing lipid, differentiated early (10th-15th postnatal days) from fibroblast-like cells of the multilayered tubule wall and later (15th-25th postnatal days) from dividing differentiated and semi-differentiated Leydig cells within the lymphatic space; fibroblastic cells arose from cells with similar morphological characteristics; semi-differentiated Leydig cells divided, and differentiated Leydig cells in the lymphatic space self-renewed; undifferentiated perivascular cells most likely gave rise to Leydig cells, pericytes; arteriolar smooth muscle cells and vascular endothelial cells arose from division of the pre-existing respective cell types. Fetal Leydig cells appeared to remain but, with time, they appeared to lose their lipid. The data suggest that (1) early recruitment of Leydig cells from undifferentiated peritubular fibroblast-like cells, (2) later mitosis of differentiated and semi-differentiated Leydig cells primarily in the interstitium but also in the perivascular region, and (3) the continued presence of pre-existing Leydig cells from the fetus constitute the adult population. Leydig cell division in the adult mouse was documented. This study provides the necessary information for the recognition of cell divisions to study of cell lineages among testis cells.

Morphometry of rat germ cells during spermatogenesis.

BACKGROUND: There has never been a study of the components of germ cells as they progress through spermatogenesis. METHODS: The structural changes taking place in rat germ cells, from spermatogonia to late spermatids, were studied utilizing morphometric techniques conducted largely at the ultrastructural level. RESULTS: Volume and surface area parameters for virtually all cellular and subcellular features were obtained for nine periods during the spermatogenic cycle. Virtually all germ cell components show dynamic properties associated with specific phases of their development. CONCLUSIONS: The data provided can be used in an objective way to characterize structural changes taking place during spermatogenesis and to relate those structural changes to functional properties of germ cells.

Sertoli cells in testes containing or lacking germ cells: a comparative study of paracrine effects using the W (c-kit) gene mutant mouse model.

BACKGROUND: Paracrine effects of germ cells on Sertoli cell structure were examined in a mouse model with the W locus (dominant white spotting) mutation in which animals with the W/Wv genotype (referred to as mutants) lack virtually all germ cells. RESULTS: Morphometric determination of Sertoli cell parameters in mutant and control (+/+) animals showed that although the testes of mutant animals were about eight times smaller than controls, the numbers of Sertoli cells in the two groups did not differ. Sertoli cell volume, Sertoli cell cytoplasmic and nuclear volumes, and Sertoli cell surface area in mutant animals were significantly smaller than in control animals. Organelle volumes and surface areas, expressed per cell, did not differ significantly in the two groups with one exception: the volume and surface area of smooth endoplasmic reticulum was significantly reduced in mutant animals. Plasma testosterone levels and tissue testosterone levels/testis were normal, indicating that the effects observed in the mutant animal were not a consequence of androgen insufficiency. Plasma FSH was elevated, probably as a consequence of germ cell depletion, and was thought not to affect Sertoli cell parameters observed. CONCLUSIONS: The data suggest that paracrine interactions with germ cells do affect Sertoli cells by modifying the amount of smooth endoplasmic reticulum. These data focus attention on the function of this abundant Sertoli cell organelle in promoting spermatogenesis.

The urethral glands of male mice in relation to depletion of secretory granules upon mating.

The present study describes the effects of mating on urethral gland acinar cells in male mice. Histological and morphometric analysis demonstrated that there was a depletion of secretory granules in the urethral glands during mating. However, no change occurred in the rough endoplasmic reticulum containing tubular elements. The results indicate that the urethral glands are functional during mating. The timing of their granule depletion suggests that urethral gland secretions may contribute to the formation of semen or the copulation plug.

Hormone sensitivity of germ cells in stage XIV and in stage I of the rat spermatogenic cycle.

Previous data have been inconclusive with respect to whether the meiotic degenerations that occur in stage XIV of the spermatogenic cycle are increased after hypophysectomy. Meiotic cell degenerations in Stage XIV and early Stage I of the spermatogenic cycle were enumerated to determine if the advanced generation of meiotic cells were influenced by hormonal deprivation subsequent to hypophysectomy and, if so, could cellular degenerations be prevented by supplementation with either testosterone or recombinant FSH during the period of hypophysectomy. The animals utilized were either pituitary-intact rats or rats hypophysectomized for 3 or 10 days. Hormone supplementation began at day 3 post-hypophysectomy and continued until day 10 at which time all animals were sacrificed. The numbers of degenerating meiotic figures (metaphase to telophase of the first and second meiotic division) as expressed per Sertoli cell nucleus or nucleolus were not increased significantly 10 days after hypophysectomy as compared with animals hypophysectomized for 3 days or with pituitary-intact controls. Exogenously administered testosterone and FSH had no effect on the numbers of degenerating meiotic germ cells in hypophysectomized animals. These data indicate that stage XIV metaphase to telophase spermatocytes are not hormone sensitive. However, it was determined that there were new cell types degenerating at Stage XIV and I of the spermatogenic cycle. These were interphase secondary spermatocytes and step 1 spermatids and were seen in stages XIV and I, respectively. These cell degenerations were found in low numbers in Stage XIV and I in either FSH-treated or testosterone-treated rats, suggesting their hormone sensitivity. Thus it is possible that Stages XIV and I are hormone sensitive stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface and surface-to-volume relationships of the Sertoli cell during the cycle of the seminiferous epithelium in the rat.

The surface relationships of the Sertoli cell and the surface relationships of the Sertoli cell in comparison to the changing volumes of developing germ cells were studied using morphometric techniques at periods representing nine groupings of the fourteen defined periods in the cycle of the seminiferous epithelium of the adult rat. No cyclic variation in the total Sertoli plasma membrane surface area was noted. Cyclic variations were noted in the area of the Sertoli cell surface that faces the basal compartment germ cells, but not the basal lamina. No cyclic variations were noted in the amount of contact of the Sertoli cells with each other at the level of the Sertoli cell barrier. However, when areas in the adluminal compartment were studied, significantly less Sertoli-Sertoli contact was seen in stages V through VII than in other stages with the exception of stages II-IV. Surface contact of germ cells with Sertoli cells increased progressively as germ cells entered the intermediate compartment and progressed to late spermatids. However, a calculation of the surface-to-volume ratio showed that surface increases of the Sertoli cell in relation to the volume of germ cells were greatest in elongating spermatids past step 12 of spermiogenesis. The area in which Sertoli ectoplasmic specializations faced germ cells was determined throughout spermatogenesis, and these data demonstrated that the first appearance of ectoplasmic specialization was at the mid-pachytene phase. They also showed that stage VIII was a period when ectoplasmic specialization loss from the cell surface was evident. Less Sertoli ectoplasmic specialization face step 8 and step 19 spermatids than comparable germ cell types at other stages. In addition to Sertoli cell surface area changes during the cycle, volumes of individual germ cell types were determined for the first time. The data presented allow an objective understanding of the complex structure and relationships of the Sertoli cell and provide a basis for understanding functional changes and interpreting biochemical data.

Recombinant human follicle-stimulating hormone is capable of exerting a biological effect in the adult hypophysectomized rat by reducing the numbers of degenerating germ cells.

There is considerable controversy as to whether FSH can, under normal circumstances, exert an effect to promote spermatogenesis in the adult rat. Recombinant human FSH (rhFSH) was used to answer a more limited question relating to whether FSH is capable of exerting a biological effect in promoting adult spermatogenesis. Can a pure preparation of FSH prevent the regressive changes seen after hypophysectomy (Hx) in a short term experiment? To answer this question, five groups of adult rats were used as follows: pituitary-intact animals, 3-day hypophysectomized (Hx), 3-day Hx given 3 mg testosterone propionate (T)/day for 7 days, 3-day Hx given 22 IU rhFSH for 7 days, and 3-day Hx given saline vehicle for 7 days. Testis weight, seminiferous tubule diameter, analysis of four degenerating germ cell types, the relative amount of lipid, and the levels of FSH receptors showed that FSH could, in a significant manner, prevent the regressive changes accompanying Hx. FSH was not as effective as T in doing so, because the FSH values were always intermediate between T-maintained animals and those after long term Hx. The Leydig cell was eliminated as a possible source of FSH-stimulated T promotion of spermatogenesis, given that morphometry and tissue T assays indicated that no additional production of T was elicited by rhFSH. The assay system used to enumerate degenerating germ cells proved a very sensitive indicator of the ability of hormones to maintain cell viability in short term experiments. The data not only show that FSH can exert a biological effect, but that this effect is qualitatively similar to that seen after the administration of T in terms of the maintenance of viability of specific germ cell types. A hypothesis is presented whereby FSH and T, although the former acting by a second messenger system and the latter by binding to nuclear receptors, can stimulate the genome to exert similar qualitative effects promoting the viability of germ cells.

Sertoli cell cycle: a re-examination of the structural changes during the cycle of the seminiferous epithelium of the rat.

Seminiferous tubules in mammals are composed of cell associations that show a cyclic pattern of renewal and development. The cyclic nature of germ cell development suggests that the cells supporting the spermatogenic process, the Sertoli cells, might also differ structurally during the spermatogenic cycle in terms of the quantity of their constituents. In the present study, cyclic differences in volumes, and surface areas were determined using a sampling technique at the electron microscope level that proportionally samples the Sertoli cell within the seminiferous tubule. Among the many parameters studied, only the surface area of the cell, the volume of lipid, and the volume and surface area of the rough endoplasm reticulum were shown by statistical analysis to vary cyclically. Regarding rough endoplasm reticulum, the volume and surface area of this organelle peaked at mid-cycle and its low was recorded near the end of the cycle, exhibiting an approximate 15-fold difference between extremes. The rough endoplasm reticulum parameters generally correlated with known patterns of protein secretion within the tubule and with the secretion of specific proteins as well as the factors important in controlling protein secretion. Many Sertoli cell structural parameters suggested to be influenced cyclically in the rat in other studies could not be confirmed by the present study. Methodological differences in the present study and past studies are discussed as potential sources of error for these discrepancies.