Protein malnutrition during critical periods poses significant risks to reproductive health. Thus, this study aims to evaluate the immediate and delayed effects of a 30-day low-protein diet on the postnatal development of the male reproductive system. For so, male rats were fed a protein-deficient diet from postnatal day 30–60, followed by evaluations of testis, epididymis, and spermatozoa both at the end of the diet and after a 60-day recovery period. Testicular and epididymal weight was lowered in pubertal animals. Several histological alterations were found in the testis, such as acidophilic cells and vacuoles in the seminiferous epithelium, and sperm production was compromised. In the epididymis, the luminal compartment was diminished, and the stroma was enlarged both in the caput and cauda; in the cauda, the epithelial compartment was enlarged; the transit time of spermatozoa through this organ was diminished. Testosterone production was lowered. Spermatozoa’s motility, mitochondrial activation, and acrosomal integrity were impaired, and several alterations in morphology were observed. After the recovery period, testicular and epididymal weight was restored. Tissue remodulation was observed in the epididymis, but the spermatozoa’s transit time in this organ was not altered. Sperm and testosterone production, spermatozoa motility, mitochondrial activation, and acrosomal integrity were also restored. However, testicular histological alterations and spermatic morphological abnormalities were maintained in protein-restricted animals. Protein restriction during peripuberty impairs the reproductive maturation of pubertal Wistar rats, impairing testicular and epididymal function, with lasting effects even after dietary correction.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood–testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

Japanese fancy mouse, mini mouse or pet mouse are common names used to refer to strains of mice that present with different colour varieties and coat types. Although many genetic studies that involve spotting phenotype based on the coat have been performed in these mice, there are no reports of quantitative data in the literature regarding testis structure and spermatogenic efficiency. Hence, in this study we researched testis function and spermatogenesis in the adult Japanese fancy mouse. The following values of 68 ± 6 mg and 0.94 ± 0.1% were obtained as mean testis weight and gonadosomatic index, respectively. In comparison with other investigated mice strains, the fancy mouse Leydig cell individual size was much smaller, resulting in higher numbers of these cells per gram of testis. As found for laboratory mice strains, as a result of the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle have been described in this study. The combined frequencies of pre-meiotic and post-meiotic stages were respectively 24% and 64% and very similar to the laboratory mice. The more differentiated germ cell types marked at 1 h or 9 days after tritiated thymidine administration were preleptotene/leptotene and pachytene spermatocytes at the same stage (VIII). The mean duration of one spermatogenic cycle was 8.8 ± 0.01 days and the total length of spermatogenesis lasted 37.8 ± 0.01 days (4.5 cycles). A high number of germ cell apoptosis was evident during meiosis, resulting in lower Sertoli cell and spermatogenic efficiencies, when compared with laboratory mice strains.

Maternal protein restriction (MPR) during pregnancy impaired the reproduction of male offspring. We investigated, during the first wave of spermatogenesis, whether MPR exerts deleterious effects on germ cell proliferation and differentiation, as well as androgen receptor (AR) protein expression, which was used as a marker for Sertoli cell (SC) maturation. At the beginning of pregnancy (day 0), dams were fed a control diet (C: 20% casein) or a restricted isocaloric diet (R: 10% casein). After birth, four groups were established: CC, RR, CR and RC (first letter diet during pregnancy and second during lactation). Male offspring were studied at postnatal days 14, 21 and 36. At birth, pup body weight was unchanged. Body weight and testis weight were reduced in RR and CR groups at all ages evaluated. MPR delayed the germinal epithelium development at all ages evaluated. On performing Western blot and immunohistochemistry, AR expression was found to be lower in the three restricted groups. The results suggest that MPR during pregnancy and/or lactation delays SC maturation and germ cell differentiation, and affects intratubular organization. These changes might be responsible for the lower fertility rate at older ages.

The cururu stingray Potamotrygon cf. histrix, a new and endemic Amazonian freshwater species, presents appropriate characteristics for fish keeping and is exploited from its natural environment. The present study identified the testicular structure and spermatogenesis of this species. Gonads from adult male specimens were dissected, fixed and processed for histological analysis. The testes were of testicular/epigonial type. The presence of germinal papillae was observed in the upper portion of organ with primordial germ cells and Sertoli cell precursors. The testis was lobular with zonal organization and cystic gametogenesis, with the occurrence of spermatoblasts. The Sertoli cells underwent morphological modifications over the course of gamete formation. The spermatozoids had long heads and were spiraled on their own axis. Information on the reproductive biology will serve as basis for studies on the reproduction and phylogeny of this peculiar group of cartilaginous fish.

Sertoli cells are very important to spermatogenesis homeostasis because they control germ cell proliferation, differentiation, and death. Damages to Sertoli cells cause germ cell death and affect fertility. Etoposide is a potent chemotherapeutic drug largely used against a variety of cancers. However, this drug also kills normal cells, especially those undergoing rapid proliferation. In the testis, etoposide acts predominantly on intermediate and type B spermatogonia. Etoposide was shown to permanently alter Sertoli cell function when administered to prepubertal rats. Based on this, we decided to investigate whether etoposide can affect Sertoli cell morphology. For this, 25-day-old rats were treated with etoposide during 8 consecutive days and killed at 32, 45, 64, 127, and 180 days old. Testes were fixed in Bouin's liquid or in a mixture of 2.5% glutaraldehyde and 2% formaldehyde for analysis under light and electron microscopes, respectively. Sertoli cells showed morphological alterations such as the presence of chromatin clumps close to the nuclear membrane, nucleus displacement, and cytoplasmic vacuolization. Some Sertoli cells also showed nuclear and cytoplasmic degenerative characteristics, suggesting that etoposide causes severe damages to Sertoli cell.