Three-dimensional analysis of seminiferous tubules and spermatogenesis in the musk shrew, Suncus murinus.

Although evidence of the spermatogenic wave in humans has been reported, little is known about the architectural arrangement of stages along the length of tubules in men with varied rates of sperm production. To conduct these studies, methodology is needed to stage relatively long lengths of straight portions of tubules in several men. Testes obtained at autopsy were glutaraldehyde perfused. Six men representing a range of daily sperm production were selected for this study. Wedges composed of groups of seminiferous tubules attached to the rete testis and separated by testicular septa were teased apart from the remaining testicular tissue, fixed in osmium, and oriented during Epon embedding. This orientation allowed for sections to be cut perpendicular to the length of the group of tubules, beginning at the end near where the tubules had been attached to the rete testis. Also, a tubule isolated away from the rete testis was oriented for perpendicular sectioning for one of the men. Several serial sections were cut at a setting of 22 microns, and each was optically sectioned approximately at 7 microns and photographed using Nomarski optics. Because many cytoplasmic and nuclear features of spermatogonia, spermatocytes, and spermatids were distinguished in photomicrographs produced by Nomarski optics, this optical system facilitated the identification of stages of the spermatogenic cycle in human seminiferous tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel coat-color mutant was found in the musk shrew (Suncus murinus). Mutant shrews were characterized by light-gray coat, pinkish skin and red eyes. Mating experiment demonstrated that the mutant character was controlled by a single autosomal recessive gene. The gene could be traced back to at least four heterozygous carriers captured in Naha city, Okinawa in 1983. The name, red-eyed dilution, was proposed for this mutant character with the gene symbol rd. Linkage analysis proved no close relationship of the rd locus with the cr (cream coat color) and ch (curly hair) loci. The red-eyed dilution shrews (+/+, rd/rd) could easily be distinguished from the cream coat shrews with dark-red eyes (cr/cr, +/+) and the double homozygotes exhibiting light-cream coat with pink eyes (cr/cr, rd/rd). The rd gene has been maintained in the OKI line about at 75% of its frequency in every generation. We have started to develop a new line triple-homozygous for the cr, ch and rd genes.

Simultaneous production of gonadotrophins (FSH/LH) and thyrotrophin (TSH) in single anterior pituitary cells of the musk shrew (Suncus murinus)

The distribution of F-actin, vimentin and alpha-tubulin was studied immunohistochemically in bovine seminiferous and straight testicular tubules, rete testis and intertubular tissue during postnatal development. Sites of antigenicity were detected by ABC immunoperoxidase technique and visualized by metal-enhanced deposition of diaminobenzidine. Within the seminiferous epithelium, F-actin appears at 20 weeks and is found in adult Sertoli cells as part of specialized cell contacts. In peritubular cells, F-actin increases gradually from 4 to 30 weeks when the adult concentration is achieved. After 20 weeks, subepithelial fibroblasts of the mediastinum testis start to express F-actin and at 52 weeks, a thick layer of positive myofibroblasts is seen beneath the epithelia of rete testis and straight testicular tubules. Testicular macrophages and light intercalated cells (LIC) are also characteristically decorated following F-actin immunoreaction. Vimentin is localized in perinuclear position in pre-Sertoli cells of 4-20 weeks and in adult Sertoli cells. During the period of transformation from pre-Sertoli to Sertoli cells, the perinuclear vimentin coat is absent. The epithelia of rete testis and straight tubules exhibit a strong vimentin immunoreaction in their basal parts. This specific pattern does not change from 4 weeks to adulthood. Alpha -tubulin is absent in 4-week-old seminiferous tubules. At 8 weeks, the perinuclear area of pre-Sertoli cells reacts positive. The alpha-tubulin content increases in these cells continuously, and from 30 weeks on nearly the entire supranuclear cytoplasm of Sertoli cells is heavily decorated. The epithelial of rete and straight tubules display a growing number of alpha-tubulin-positive cells from 4 to 40 weeks. From then on, nearly all epithelial cells contain alpha-tubulin, particularly in a narrow zone beneath their lateral cell borders.

To describe pubertal testicular growth in humans, changes in testicular cell populations that result in testicular growth, and the role of testosterone and gonadotrophins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in testicular growth. When human data were not available, studies in nonhuman primates and/or rodents were used as surrogates. Testicular growth in puberty follows a sigmoidal growth curve, with a large variation in timing of testicular growth and adult testicular volume. Testicular growth early in puberty is due to increase in Sertoli cell number and length of seminiferous tubules, whereas the largest and fastest growth results from the increase in the diameter of the seminiferous tubules first due to spermatogonial proliferation and then due to the expansion of meiotic and haploid germ cells. FSH stimulates Sertoli cell and spermatogonial proliferation, whereas LH/testosterone is mandatory to complete spermatogenesis. However, FSH and LH/testosterone work in synergy and are both needed for normal spermatogenesis. Testicular growth during puberty is rapid, and mostly due to germ cell expansion and growth in seminiferous tubule diameter triggered by androgens. Pre-treatment with FSH before the induction of puberty may improve the treatment of hypogonadotropic hypogonadism, but remains to be proven.

Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glycemic control and promote weight loss in diabetes and obesity but are also associated with gastrointestinal adverse events, including nausea and emesis in many patients. These concerns highlight the need for the development of novel GLP-1R agonists that minimize these side effects while maintaining beneficial metabolic outcomes. Here, we investigate the in vivo effects of exendin-4-Phe1 (Ex-Phe1), a GLP-1R biased agonist. In three pre-clinical species, mice (n = 43), rats (n = 54), and musk shrews (n = 30), we examined in vivo glycemic control, feeding, and nausea/emesis following native Ex-4 and Ex-Phe1 administration. We also used cFos expression following Ex-4 and Ex-Phe1 administration to examine neural activation in regions involved in mediating nausea and emetic side effects of GLP-1R agonism. In vitro studies show Ex-Phe1 favors cAMP signaling with reduced β-arrestin recruitment. Compared to Ex-4, Ex-Phe1 produced fewer emetic episodes in musk shrews (Suncus murinus) and little to no pica, a proxy for nausea, in rats. Ex-Phe1 effects on food intake and body weight varied by species, while Ex-4 and Ex-Phe1 similarly enhanced glucose tolerance in all species. Ex-4 and Ex-Phe1 increased cFos expression within brain regions linked to nausea and emesis in all species. Collectively, Ex-Phe1 maintains glycemic benefits in all three species, but putatively blunts the ability of the CNS GLP-1R+ cells to drive anorexia and weight loss, as well as unwanted adverse events (nausea/emesis) in rats and musk shrews.

Several cancer chemotherapies cause nausea and vomiting, which can be dose-limiting. Musk shrews are used as preclinical models for chemotherapy-induced emesis and for antiemetic effectiveness. Unlike rats and mice, shrews possess a vomiting reflex and demonstrate an emetic profile similar to humans, including acute and delayed phases. As with most animals, dosing of shrews is based on body weight, while translation of such doses to clinically equivalent exposure requires doses based on body surface area. In the current study body surface area in musk shrews was directly assessed to determine the Meeh constant (Km) conversion factor (female = 9.97, male = 9.10), allowing estimation of body surface area based on body weight. These parameters can be used to determine dosing strategies for shrew studies that model human drug exposures, particularly for investigating the emetic liability of cancer chemotherapeutic agents.

Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-μm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid-Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells. In brief: Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells.

Oxidation of Fatty Acids by Kidney Microsomes of Musk Shrew (Suncus murinus)

The musk shrew, Suncus murinus, is a unique animal model for the study of female behavioral endocrinology, and by utilizing the physiological characteristics of ovulation during mating of S. murinus, key progress has been made in the study of the endocrine regulatory function of the ovary. The study of innervation of the ovary of S. murinus is particularly important. In the present study, the 3-dimensional structure of the neural innervation of the internal genital organs of S. murinus (n=11, female) was studied using whole-mount immunohistochemical staining with neurofilament protein antibodies. The innervation of the ovary in S. murinus contains the superior ovarian nerve, which runs along the suspensory ligament of the ovary, and the ovarian plexus nerve, which runs along the ovarian artery and vein. The innervation of the uterus originates from the lower hypogastric plexus running in the pelvic peritoneum and along the uterine artery and vein. There was abundant communication between the ovarian plexus nerve and the lower hypogastric plexus of the pelvic peritoneum. The present study is the first to visualize the (NFP-positive) innervation of the female internal genital organs of the experimental animal S. murinus by the whole-mount immunohistochemistry method. The abundant communication between the ovarian plexus nerve and the lower hypogastric plexus of the pelvic peritoneum provides an additional pathway for the neural regulation of the ovary.

92008 ?? 33 1214 Received 2.8.2007 ; a cepted 5.5.2008 Copyright © 8 MSJ Introduction The ho s musk shrew (Suncus murinus) is one of 15 species in the genus Suncus (Abe 1985). This species is widely distributed from the Middle East and the Indian subcontinent to Southeast Asia, from tropical to subtropical regions, and its northernmost recorded habitat is in Nagasaki Prefecture, Japan (Corbet and Hill 1992). In Japan, the house musk shrew has been an established laboratory animal since 1973, and several laboratory strains currently exist (e.g., KAT, NAG, RYU, TESS; Oda 1991). This is the only widely used laboratory animal belonging to the Insectivora; suncus is utilized for a variety of purposes, including emesis studies (Ueno et al. 1987; Uchino et al. 2006). The house musk shrew exhibits extreme morphological variations. For example, its body weight varies threeto fourfold among wild populations: male shrews in Bangladesh weigh more than 150 g, while males in Guam are less than 50 g (Tomita et al. 1984; Ishikawa et al. 1989). Variations also exist in the dentition (Hanamura et al. 1979; Jogahara et al. 2007), chromosome number (Minesawa 1985), and coat color (Iseki et al. 1984; Ishikawa et al. 1986, 1987, 1989). Moreover, mutations in coat color (cream and white) and eye color (red-eyed dilution) occur in wild populations (Oda and Shigehara 1978; Ohno et al. 1992, 1994). The most prominent form of albinism in mice is a tyrosinase gene mutation that results in a white coat and red eyes (Beermann et al. 2004). However, mutations in other pigment genes result in diverse phenotypes in mice and humans (Tomita and Suzuki 2004; Hearing 2005). Thus, phenotypic observations cannot determine if the tyrosinase gene mutation is present. Moreover, individuals with a white coat and normal eye color occur in many species, including the house musk shrew (Oda and Shigehara 1978). Hence, an albino-like shrew is defined as one having a pure white coat and red eyes (Fig. 1a). Three albino-like house musk shrews were captured on Okinawa Island, Japan. In this report, past capture records of albino-like house musk shrews in the Ryukyu Archipelago were reviewed and integrated present capture records to determine their distribution. Additionally, the captured albino-like house musk shrews were maintained in the laboratory, and bred with the RYU strain of suncus established from Okinawa Island. The RYU strain had been kept for over 4 years and over 60 litters have been obtained, although no record exists of coat color variation or albinism and red eyes. This is thus a report of preliminary findings on the inheritance of the albino-like gene. Five albino-like house musk shrews from Okinawa and Tarama Islands have been recorded since 1968 (Table 1). Although the house musk shrew is a common

Asian musk shrews (Suncus murinus) are induced ovulators, but exhibit no cyclic changes in reproductive structures or in sexual behavior. Mating behavior is induced by contact with a male. To determine if mating induces changes in ovarian steroidogenesis, ovaries removed from unmated animals and at 3, 10, 15, and 36 h after mating were cultured for 4 h in the presence or absence of gonadotropins (LH + FSH, 1 microgram/ml). Histological analysis revealed no obvious changes in follicular size or appearance at the end of culture in ovaries cultured at 3 and 10 h post-mating, as compared with ovaries from unmated shrews, and mating did not stimulate any discernable changes in steroid secretion in these two groups. However, at the end of the culture period, ovulation had occurred or was occurring in ovaries from 35% of the animals ovariectomized at 15 h after mating, and corpora lutea (CLs) were present in 39% of ovarian pairs obtained 36 h after mating. At 15 h post-mating, ovaries with ovulations secreted three times more estradiol than did ovaries that showed no evidence of stimulation by mating, but there were no differences in testosterone or progesterone production. In contrast, ovaries isolated 36 h post-mating with CLs secreted dramatically more of all three steroids than ovaries without CLs (23, 13, and 52 times more estradiol, testosterone, and progesterone, respectively). These data are consistent with plasma concentrations of estradiol at the time of ovariectomy, which were twice as high at both 15 and 36 h after mating, in animals whose ovaries showed evidence of ovarian stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of sexual receptivity in the female musk shrew ( Suncus murinus)

The effects of exogenous testosterone on various testicular variables has become of increasing significance because of its potential use in male contraception. For this reason, high doses of two testosterone esters [testosterone propionate (TP) and testosterone enanthate (TE)] were used in a study of their influence on the morphology, length and curvature of the seminiferous tubules of the rat testis, and on cytological smears of the seminiferous tubules epithelium. TP was given for 14 days (3 mg/100 g body weight, i.m.) to assess the acute effects of testosterone on the seminiferous tubules. TE was administered for 60 days (in the same manner as TP) to study possible chronic effects on the rat testis. After TP and TE treatment the seminiferous tubule epithelium showed disorganization and desquamation of spermatogenic cells. In the TP-treated testes the tubules lined with Sertoli cells only were observed. The values for the length and curvature of seminiferous tubules of the TP- and TE-treated rats were significantly reduced (p < 0.001). All these changes were observed earlier in the TP-treated than in the TE-treated animals. In cytological smears of the testis of the TP- and TE-treated rats an increase of vacuoles and residual bodies in Sertoli cell cytoplasm was noted. In addition, a reduction of spermatogenic cells, particularly sperms, was manifest in the smears after treatment. Large groups of Sertoli cells were seen in the smears from these testes.

Evaluation of changes in the secretion of immunoactive inhibin by adult rat seminiferous tubules in vitro as an indicator of early toxicant action on spermatogenesis