Environmental factors in declining human fertility.

A severe decline in child births has occurred over the past half century, which will lead to considerable population declines, particularly in industrialized regions. A crucial question is whether this decline can be explained by economic and behavioural factors alone, as suggested by demographic reports, or to what degree biological factors are also involved. Here, we discuss data suggesting that human reproductive health is deteriorating in industrialized regions. Widespread infertility and the need for assisted reproduction due to poor semen quality and/or oocyte failure are now major health issues. Other indicators of declining reproductive health include a worldwide increasing incidence in testicular cancer among young men and alterations in twinning frequency. There is also evidence of a parallel decline in rates of legal abortions, revealing a deterioration in total conception rates. Subtle alterations in fertility rates were already visible around 1900, and most industrialized regions now have rates below levels required to sustain their populations. We hypothesize that these reproductive health problems are partially linked to increasing human exposures to chemicals originating directly or indirectly from fossil fuels. If the current infertility epidemic is indeed linked to such exposures, decisive regulatory action underpinned by unconventional, interdisciplinary research collaborations will be needed to reverse the trends.

Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV.

Viruses have colonized the germ line of our ancestors on several occasions during evolution, leading to the integration in the human genome of viral sequences from over 30 retroviral groups and a few nonretroviruses. Among the recently emerged viruses infecting humans, several target the testis (e.g., human immunodeficiency virus [HIV], Zika virus, and Ebola virus). Here, we aimed to investigate whether human testicular germ cells (TGCs) can support integration by HIV, a contemporary retrovirus that started to spread in the human population during the last century. We report that albeit alternative receptors enabled HIV-1 binding to TGCs, HIV virions failed to infect TGCs in vitro Nevertheless, exposure of TGCs to infected lymphocytes, naturally present in the testis from HIV+ men, led to HIV-1 entry, integration, and early protein expression. Similarly, cell-associated infection or bypassing viral entry led to HIV-1 integration in a spermatogonial cell line. Using DNAscope, HIV-1 and simian immunodeficiency virus (SIV) DNA were detected within a few TGCs in the testis from one infected patient, one rhesus macaque, and one African green monkey in vivo Molecular landscape analysis revealed that early TGCs were enriched in HIV early cofactors up to integration and had overall low antiviral defenses compared with testicular macrophages and Sertoli cells. In conclusion, our study reveals that TGCs can support the entry and integration of HIV upon cell-associated infection. This could represent a way for this contemporary virus to integrate into our germ line and become endogenous in the future, as happened during human evolution for a number of viruses.IMPORTANCE Viruses have colonized the host germ line on many occasions during evolution to eventually become endogenous. Here, we aimed at investigating whether human testicular germ cells (TGCs) can support such viral invasion by studying HIV interactions with TGCs in vitro Our results indicate that isolated primary TGCs express alternative HIV-1 receptors, allowing virion binding but not entry. However, HIV-1 entered and integrated into TGCs upon cell-associated infection and produced low levels of viral proteins. In vivo, HIV-1 and SIV DNA was detected in a few TGCs. Molecular landscape analysis showed that TGCs have overall weak antiviral defenses. Altogether, our results indicate that human TGCs can support HIV-1 early replication, including integration, suggesting potential for endogenization in future generations.

Spermatogonial stem cell markers and niche in equids.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called "niche" that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions.

Endocrine modulation of the recipient environment affects development of bovine testis tissue ectopically grafted in mice.

Testis tissue xenografting is a powerful approach for the study of testis development and spermatogenesis, and for fertility preservation in immature individuals. In bovine testis xenografts, maturation and spermatogenesis are inefficient when compared to other species. To evaluate if exogenous modulation of the endocrine milieu in recipient mice will affect spermatogenic efficiency in xenografts from newborn calves, recipient mice were treated with the GnRH antagonist acyline (5 mg/kg s.c. every 2 weeks) to reduce testosterone production in xenografts, or with 6-N-propyl-2-thiouracil (PTU, 0.1% in drinking water for 4 weeks), to induce transient hypothyroidism in recipient mice respectively. Both treatments altered developmental parameters of testis xenografts and reduced germ cell differentiation. While the effects of acyline treatment can be attributed to inhibition of GnRH and gonadotropin action, lower Sertoli cell numbers and decreased seminiferous tubule length observed after PTU treatment were opposite to effects reported previously in rats. Regardless of treatment, Sertoli cells underwent only partial maturation in xenografts as Müllerian inhibiting substance and androgen receptor expression were lower than in donor and adult tissue controls respectively. In conclusion, although treatments did not result in improvement of maturation of bovine testis xenografts, the current study demonstrates that exogenous modulation of the endocrine milieu to affect xenograft development in recipient mice provides an accessible model to study endocrine control of spermatogenesis in large donor species.

Sertoli cell proliferation in the adult testis is induced by unilateral gonadectomy in African catfish.

Survival and development of male germ cells depends on their close contact with Sertoli cells. In the cystic spermatogenesis found in fish, one germ cell clone, initially a single undifferentiated spermatogonium type A, is enclosed by and accompanied through spermatogenesis by a group of Sertoli cells. Previous work showed that after forming such spermatogenic cysts, Sertoli cells proliferated mainly during the mitotic expansion of the spermatogonial clone in the cyst. Here, we used unilateral gonadectomy (ULG) as experimental model to study Sertoli cell proliferation at the start of cyst development in adult African catfish testis. Four days after surgery, we observed a particularly strong increase in the number of mitotic Sertoli cells along with a significant increase in the number of mitotic single type A spermatogonia. Proliferation of pairs of spermatogonia or of larger germ cell clones, however, did not change. At the same time, pituitary transcript levels of the three gonadotropin-subunits (cga, glycoprotein hormones, alpha polypeptide; fshb, follicle stimulating hormone, beta polypeptide; lhb, luteinizing hormone, beta polypeptide) were not different between sham-operated and ULG males. However, expression of the gonadotropin-releasing hormone receptor gene gnrhr1 was significantly reduced after ULG, and Lh plasma levels were slightly elevated. In the testis remaining after ULG, Fsh receptor (fshr) mRNA levels increased significantly but luteinizing hormone/choriogonadotropin receptor (lhcgr) mRNA levels did not change. Circulating androgen levels did not differ between groups, but testicular androgen release increased significantly 2- to 3-fold after ULG. Considering the strong steroidogenic potency of Fsh and the expression of the fshr gene by Leydig cells in catfish, we explain the absence of an effect of ULG on circulating androgen levels by an Fshr-mediated, compensatory increase in the steroid production of the remaining testis, perhaps supported in addition by the increased Lh plasma levels. Since Fsh is a major stimulator of mammalian Sertoli cell proliferation, we propose that ULG-induced activation of the Fsh signalling system also promoted Sertoli cell proliferation and - possibly as a consequence of that - proliferation of single type A spermatogonia, providing the basis for an increased spermatogenic capacity.

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu).

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.

Germ cell transplantation in felids: a potential approach to preserving endangered species.

With the exception of the domestic cat, all members of the family Felidae are considered either endangered or threatened. Although not yet used for this purpose, spermatogonial stem cell (SSC) transplantation has a high potential to preserve the genetic stock of endangered species. However, this technique has not previously been established in felids. Therefore, we developed the necessary procedures to perform syngeneic and xenogeneic SSC transplants (eg, germ cell [GC] depletion in the recipient domestic cats, enrichment and labeling of donor cell suspension, and the transplantation method) in order to investigate the feasibility of the domestic cat as a recipient for the preservation and propagation of male germ plasm from wild felids. In comparison with busulfan treatment, local x-ray fractionated radiation was a more effective approach to depleting endogenous spermatogenesis. The results of both syngeneic and xenogeneic transplants revealed that SSCs were able to successfully colonize and differentiate in the recipient testis, generating elongated spermatids several weeks posttransplantation. Specifically, ocelot spermatozoa were observed in the cat epididymis 13 weeks following transplantation. As donor GCs from domestic cats and ocelots were able to develop and form mature GCs in the recipient environment seminiferous tubules, these findings indicate that the domestic cat is a suitable recipient for SSC transplantation. Moreover, as modern cats descended from a medium-size cat that existed approximately 10 to 11 million years ago, these results strongly suggest that the domestic cat could be potentially used as a recipient for generating and propagating the genome of wild felids.

A new and fast technique to generate offspring after germ cells transplantation in adult fish: the Nile tilapia (Oreochromis niloticus) model.

BACKGROUND: Germ cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan. METHODOLOGY/PRINCIPAL FINDINGS: Using two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods. CONCLUSION: Therefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserve endangered fish species.

TGF-beta1 system in Leydig cells. Part II: TGF-beta1 and progesterone, through Smad1/5, are involved in the hyperplasia/hypertrophy of Leydig cells.

Several reports indicate that transforming growth factor beta1 (TGF-beta1) participates in the regulation of cell cycle progression. In this work, we analyzed the in vitro effect of TGF-beta1 on Leydig cell proliferation markers and the in vivo effect of this cytokine in Leydig cell hyperplasia/hypertrophy. The in vitro effect of TGF-beta1 (1 ng/ml) plus progesterone (10(-6) M) on purified Leydig cells from 3 week-old mice increased the immunocytochemically detected PCNA and stimulated the phosphorylation of Smad 1/5. Progesterone (10(-6) M) in the presence or absence of TGF-beta1 diminished the ratio Bax/Bcl-2. Morphometric testicular studies of mice treated with progesterone (s.c.) plus TGF-beta1 (intratesticular), showed an increase in interstitial volume and a decrease in tubular volume. Furthermore, the cytoplasmic volume of Leydig cells showed an increment in this experimental group with a diminution in nuclear volume. Thus, it turned out that the administration of progesterone and TGF-beta1 augmented the volume of Leydig cells. These results indicate a clear effect of TGF-beta1 in the hypertrophy/hyperplasia of Leydig cells.

Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.

The zebrafish has become an important vertebrate model for basic and biomedical research, including the research field of the biology of reproduction. However, very few morphological and stereological data are available regarding zebrafish testis structure and spermatogenesis. In this careful histomorphometric evaluation of the testis, we studied spermatogonial cells using molecular markers, determined the combined duration of meiotic and spermiogenic phases, and examined the formation of the Sertoli cell barrier (tight junctions). We found at least nine spermatogonial generations and propose a morphology-based nomenclature for spermatogonial generations that is compatible with the one used in higher vertebrates. The number of germ cells per cyst increased dramatically (1 to approximately 1360 cells) from undifferentiated spermatogonia type A to early spermatids. The combined duration of meiotic and spermiogenic phases is approximately 6 days, one of the shorter periods among the teleost fish investigated to date. The number of Sertoli cells per cyst increased 9-fold during the maturational cycle of spermatogenic cysts and stabilized in the meiotic phase at a ratio of approximately 100 early spermatids per Sertoli cell (Sertoli cell efficiency). Similarly to mammals, Sertoli cell proliferation ceased in the meiotic phase, coinciding with the formation of tight junctions between Sertoli cells. Hence, the events taking place during puberty in the germinal epithelium of mammals seem to recapitulate the "life history" of each individual spermatogenic cyst in zebrafish.

Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1.

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but have aneuploid progeny. When studying fertility in males in a two-fold more inbred background, we have however observed low numbers of fertilized eggs (approximately 0.4%). Histological examination of the testis has revealed that all spermatogenic stages prior to spermatids (spermatogonia, primary spermatocytes, and secondary spermatocytes) are significantly increased in the mutant, whereas the total weight of spermatids and spermatozoa is highly decreased (1.8 mg in wild-type vs. 0.1 mg in mutants), a result clearly different from our previous study in which outbred males lack secondary spermatocytes or postmeiotic cells. Thus, a delay of both meiotic divisions occurs rather than complete arrest during meiosis I in these males. Eggs fertilized with mutant sperm develop as malformed embryos and are aneuploid making this male phenotype much more similar to that previously described in the mutant females. Therefore, crossovers are still essential for proper meiosis, but meiotic cell divisions can progress without it, suggesting that this mutant is a suitable model for studying the cellular mechanisms of completing meiosis without crossover stabilization.

Mild calorie restriction does not affect testosterone levels and testicular gene expression in mutant mice.

The hypothalamic-pituitary-gonadal (HPG) axis and the somatotropic axis are influenced by nutritional factors. Calorie restriction (CR) extends lifespan but suppresses both the HPG and the somatotropic axes. Since most CR studies use a fairly severe (40%-60%) reduction of calorie intake, we hypothesized that a milder CR (20%) might not be deleterious to reproduction in male mice. To test this hypothesis, we evaluated the effects of 20% CR on testicular testosterone content and on testicular expression of genes that are relevant to testicular function and reproductive competence, including insulin-like growth factor-I, cytochrome P450 aromatase (Cyp19a1), androgen receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, cytochrome P450c17 and 3-beta-hydroxysteroid dehydrogenase/isomerase. To relate CR effects to the activity of the somatotropic axis, we have used growth hormone-resistant GHR knockout mice as well as transgenic mice overexpressing GH. Mild CR did not affect testosterone levels in testis homogenates and had little effect on expression of the examined genes in the reproductive organs. Altered activity of the GH/insulin-like growth factor-1 axis had a major impact on the parameters analyzed. The results also suggest that expression of several key genes involved in the control of testicular function is preserved under conditions of mild CR and encourage speculation that mild regimens of CR can produce longevity benefits without impairing reproduction.

Sertoli cell proliferation in the adult testis--evidence from two fish species belonging to different orders.

Germ cell survival and development critically depend on the cells' contact with Sertoli cells in the vertebrate testis. Fish and amphibians are different from mammals in that they show a cystic type of spermatogenesis in which a single germ cell clone is enclosed by and accompanied through the different stages of spermatogenesis by an accompanying group of Sertoli cells. We show that in maturing and adult testes from African catfish and Nile tilapia, Sertoli cell proliferation occurs primarily during spermatogonial proliferation, allowing the cyst-forming Sertoli cells to provide the increasing space required by the growing germ cell clone. In this regard, coincident with a dramatic increase in cyst volume and number of germ cells per cyst, in Nile tilapia, the number of Sertoli cells per cyst was strikingly increased from primary spermatogonia to spermatocyte cysts. In both African catfish and Nile tilapia, Sertoli cell proliferation is strongly reduced when germ cells have proceeded into meiosis, and stops in postmeiotic cysts. We conclude that Sertoli cell proliferation is the primary factor responsible for the increase in testis size and sperm production observed in teleost fish. In mammals, Sertoli cell proliferation in the adult testis is not observed under natural conditions. However, on the level of the individual spermatogenic cyst--similar to mammals--Sertoli cell proliferation ceases when germ cells have entered meiosis and when tight junctions are established between Sertoli cells. This suggests that fish are valid vertebrate models for studying Sertoli cell physiology.

Aquaporin-1 and -9 are differentially regulated by oestrogen in the efferent ductule epithelium and initial segment of the epididymis.

BACKGROUND INFORMATION: Efferent ductules reabsorb more than 90% of the rete testis fluid, a process that involves ion transporters and AQP (aquaporin) water channels. Oestrogen has been shown to modulate the expression of the ion transporters involved in this activity, but reports of AQP regulation in the male tract have been confounding. To understand better the regulation of AQP1 and AQP9, we investigated their expression in rat efferent ductules and initial segment of the epididymis after treatment with the pure antioestrogen ICI 182,780 or bilateral efferent duct ligation, or castration, followed by hormone replacement. RESULTS: Results show that AQP9 is modulated by oestrogen in the efferent ductule epithelium, but not in the initial segment of the epididymis. DHT (5alpha-dihydrotestosterone) also modulated AQP9 in efferent ductules. AQP9 was down-regulated by the antioestrogen in efferent ductules on day 45 post-treatment, which occurred before the non-ciliated cells had shown significant loss of microvilli. DHT, but not oestradiol, modulated AQP9 expression in the initial segment of the epididymis. In contrast, testosterone, DHT, oestrogen or the antioestrogen did not alter AQP1 staining, indicating constitutive expression of AQP1 in the efferent ductule epithelium. AQP1 expression was induced in peritubular cells of efferent ductules and in the initial segment of the epididymis after castration and long-term treatment with the antioestrogen. Although peritubular AQP1 staining in efferent ductules was partially reversed by the androgens, it was not reversed after any treatment in the initial segment of the epididymis. CONCLUSIONS: These results demonstrate that efferent ductules are unique in requiring both oestrogen and androgen to regulate an important mediator of fluid reabsorption, whereas the initial segment is dependent only on androgen stimulation.

Cell-cycle inhibitors p27Kip1 and p21Cip1 regulate murine Sertoli cell proliferation.

Thyroid hormone inhibits neonatal Sertoli cell proliferation and recent results have shown that thyroid hormone upregulates cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 and p21Cip1 (also known as CDKN1B and CDKN1A, respectively) in neonatal Sertoli cells. This suggests that these CDKIs, which negatively regulate the cell cycle, could be critical in Sertoli cell proliferation. Consistent with this hypothesis, mice lacking p27Kip1 develop testicular organomegaly, but Sertoli cell numbers have not been determined. Likewise, effects of loss of p21Cip1 or both p27 and p21 on Sertoli cell number and testicular development were unknown. To determine if p27 and/or p21 regulate Sertoli cell proliferation, we measured Sertoli cell proliferation at Postnatal Day 16 and testis weight, Sertoli cell number, and daily sperm production (DSP) in 4-mo-old wild-type (WT), p21 knockout (p21KO), p27 knockout (p27KO), and p27/p21 double-knockout (DBKO) mice. Testis weights were increased 27%, 42%, and 86% in adult p21KO, p27KO, and DBKO mice, respectively, compared with WT. Sertoli cell number also was increased 48%, 126%, and 126% in p21KO, p27KO, and DBKO mice, respectively, versus WT. DSP in p21KO, p27KO, and DBKO testes also showed significant increases compared with WT mice. Although DSP was increased, there were increased spermatogenic defects observed in both p27KO and DBKO mice compared with WT. These data indicate that both p27 and p21 play an inhibitory role in regulating adult Sertoli cell number such that loss of either CDKI produces primary increases in Sertoli cell number and secondary increases in DSP and testis weight. Furthermore, loss of both CDKIs causes additive effects on DSP and testis weight, suggesting a central role for these CDKIs in testis development.

Differential hormonal regulation of estrogen receptors ERalpha and ERbeta and androgen receptor expression in rat efferent ductules.

Estrogen receptors, in addition to the androgen receptor (AR), are expressed at high levels in efferent ductules of the male reproductive tract and it is now well recognized that estrogen receptor (ER) alpha is required for the maintenance of normal structure and function of the ductules. However, little is known regarding the hormonal regulation of the receptors themselves in the male. In the present study, efferent ductule ligation and castration, followed by replacement with testosterone, dihydro-testosterone (DHT) or estradiol was used to investigate the relative importance of circulating and luminal sources of steroid for the modulation of ERalpha, ERbeta and AR in rat efferent ductules. Uni- or bilateral castration and ligation did not affect the expression of ERalpha and ERbeta, but bilateral castration caused down-regulation of AR. Replacement with DHT and testosterone alone or in combination with estradiol caused the recovery of AR expression to control levels. A slight recovery of AR was also observed after estrogen replacement. ERalpha expression was decreased to nearly undetectable levels after estrogen replacement. On the other hand, ERbeta did not show evident effects following any of the treatments, suggesting a constitutive expression of this receptor. This differential modulation of the steroid hormone receptors highlights the importance of maintaining a physiological androgen-estrogen balance to regulate the structure and function of efferent ductules in the male.

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules.

BACKGROUND: The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown. METHODS: In the present study, adult male rats were treated with ICI 182,780 for 7 to 150 days, to evaluate the time-response effects of the treatment on the pattern of ERalpha, ERbeta and AR protein expression in the efferent ductules. The receptors were localized using immunohistochemistry. RESULTS: ERalpha, ERbeta and AR have distinct cellular distribution in the testis and efferent ductules. Staining for ERalpha is nearly opposite of that for ERbeta, as ERalpha shows an increase in staining intensity from proximal to distal efferent ductules, whereas ERbeta shows the reverse. Androgen receptor follows that of ERalpha. ICI 182,780 caused a gradual but dramatic decrease in ERalpha expression in the testis and efferent ductules, but no change in ERbeta and AR expression. CONCLUSIONS: The differential response of ERalpha and ERbeta proteins to ICI 182,780 indicates that these receptors are regulated by different mechanisms in the male reproductive tract.

ER function in the adult male rat: short- and long-term effects of the antiestrogen ICI 182,780 on the testis and efferent ductules, without changes in testosterone.

Male rats, 30 d old, were treated with the antiestrogen ICI 182,780 (3-150 d) to determine sequences of events leading to testicular atrophy and infertility. Plasma testosterone and LH concentrations were unchanged. ICI 182,780 induced dilation of efferent ductules as early as 3 d post treatment, and the dilation increased over time, resulting in an overall increase of 200% in tubule diameter. A gradual reduction in height of the ductule epithelium was observed; however, the microvilli height increased up to d 73 but subsequently decreased. A transient increase in lysosomes in nonciliated cells was seen from d 15 to d 100. Testicular weight increased by d 45 and seminiferous tubules were dilated by d 52. These effects on testes persisted until d 100, but on d 150 the weight decreased and severe atrophy was observed. These testicular effects were probably owing to accumulation of fluid following inhibition of reabsorption in the efferent ductules, similar to the ER-alpha knockout mouse. In agreement with this conclusion, there was a decrease in Na+-H+ exchanger-3 mRNA and protein, which is consistent with previous studies showing that ER is required for expression of Na+-H+ exchanger-3 and ultimately fluid reabsorption in the efferent ductules.

The goitrogen 6-n-propyl-2-thiouracil (PTU) given during testis development increases Sertoli and germ cell numbers per cyst in fish: the tilapia (Oreochromis niloticus) model.

The main objectives of the present study were to investigate the effects of 6-n-propyl-2-thiouracil (PTU) on Sertoli cell proliferation, germ cell number, and testis size in Nile tilapias (Oreochromis niloticus). In this regard, young fish (approximately 1 g BW and approximately 3.5 cm total in length) were treated for a period of 40 d with different concentrations (100 and 150 ppm) of PTU. The animals were killed and analyzed on d 1, 30, 40, 98, and 208 after the beginning of the treatment. On d 30 and 40 the spermatogenic process was delayed in fish treated with PTU compared with the control group. Also at these periods, treated tilapia had decreased (P < 0.05) body weight and total length. On d 98 body weight and total length had recovered in PTU-treated fish and were similar (P > 0.05) to those of the controls. However, testis weight and gonadosomatic index (testis mass/body weight) were approximately 100% higher (P < 0.05) in treated tilapia. Similarly, the area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells per testis were significantly (P < 0.05) greater in treated fish. Nevertheless, nuclear volume and individual Leydig cell volume were significantly lower (P < 0.05) in tilapia receiving PTU treatment. Compared with controls, at 208 d all parameters analyzed presented the same trend as that observed at 98 d. In general, at 98 d the different PTU concentrations used during the treatment period induced similar effects. However, at 208 d the mean values observed for several parameters were significantly higher (P < 0.05) in fish exposed to 150 ppm. Probably due to the higher density of Sertoli cells per cyst in treated tilapia, these cells presented a smaller (P < 0.05) nucleolus and a trend to decrease its support capacity (efficiency). However, the meiotic index (germ cell loss during the two meiotic divisions) was similar (P > 0.05) in the three groups of fish investigated. Remarkably, the results found in tilapia were similar to those found for rats treated with PTU. This suggests strongly that the mechanisms of control of Sertoli cell and Leydig cell proliferation seem to be preserved during vertebrate evolution.