Kyle C. Caires

Maintaining the male germline: regulation of spermatogonial stem cells

Spermatogonial stem cells (SSCs) are a self-renewing population of adult stem cells capable of producing progeny cells for sperm production throughout the life of the male. Regulation of the SSC population includes establishment and maintenance of a niche microenvironment in the seminiferous tubules of the testis. Signaling from somatic cells within the niche determines the fate of SSCs by either supporting self-renewal or initiating differentiation leading to meiotic entry and production of spermatozoa. Despite the importance of these processes, little is known about the biochemical and cellular mechanisms that govern SSC fate and identity. This review discusses research findings regarding systemic, endocrine, and local cues that stimulate somatic niche cells to produce factors that contribute to the homeostasis of SSCs in mammals. In addition to their importance for male fertility, SSCs represent a model for the investigation of adult stem cells because they can be maintained in culture, and the presence, proliferation, or loss of SSCs in a cell population can be determined with the use of a transplantation assay. Defining the mechanisms that regulate the self-renewal and differentiation of SSCs will fundamentally improve the understanding of male fertility and provide information about the regulation of adult stem cells in other tissues.

Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis.

Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

VEGFA Family Isoforms Regulate Spermatogonial Stem Cell Homeostasis in Vivo

The objective of the present study was to investigate vascular endothelial growth factor A (VEGFA) isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell-specific distribution of VEGF isoforms, receptors, and coreceptors during testis development postnatal d 1-180 suggest a nonvascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that proangiogenic isoforms of VEGF (VEGFA(164)) stimulate SSC self-renewal, whereas antiangiogenic isoforms of VEGF (VEGFA(165)b) induce differentiation of SSC. To test this hypothesis, we used transplantation to assay the stem cell activity of SSC obtained from neonatal mice treated daily from postnatal d 3-5 with 1) vehicle, 2) VEGFA(164), 3) VEGFA(165)b, 4) IgG control, 5) anti-VEGFA(164), and 6) anti-VEGFA(165)b. SSC transplantation analysis demonstrated that VEGFA(164) supports self-renewal, whereas VEGFA(165)b stimulates differentiation of mouse SSC in vivo. Gene expression analysis of SSC-associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSC. These findings indicate a nonvascular role for VEGF in testis development and suggest that a delicate balance between VEGFA(164) and VEGFA(165)b isoforms orchestrates the cell fate decisions of SSC. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis.

Endocrine regulation of spermatogonial stem cells in the seminiferous epithelium of adult mice.

Abstract A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout male life. The seminiferous epithelium is organized into stages of spermatogenesis based on the complement of germ cell types within a tubular section of the testis. The stages exist in close physical proximity and foster diverse phases of germ cell development despite exposure to a similar endocrine milieu that supports coordinated spermatogenesis. The objective of the current study was to identify the population dynamics of SSCs in vivo. We hypothesized that SSC populations and their niches are specifically distributed across the mature seminiferous epithelium in the mouse testis. To test this hypothesis, we conducted stem cell transplantation of germ cells obtained from stage-specific clusters of seminiferous tubules representing areas of high responsiveness to follicle-stimulating hormone (IX–I), androgen (II–IV), and retinoid (V–VIII) signaling. Similarly, we analyzed the expression of genes linked with SSC activity in these groups of stages. No stage-specific differences in the colonization efficiency or the colony number were detected after SSC transplantation, indicating that SSCs are equally distributed across all stages of the seminiferous tubule. In contrast, SSCs obtained from donor stages IX–IV established larger donor-derived colonies due to increased colony expansion. SSCs originating from different stages have varying degrees of stem cell activity in vivo, a notion consistent with Gdnf, Ret, and Bcl6b expression data. These results support the conclusion of a stage-specific, microenvironment-regulating SSC self-renewal and suggest the presence of a transit-amplifying population of undifferentiated spermatogonia in vivo.

Endocrine Regulation of the Establishment of Spermatogenesis in Pigs

Somatic and germ cell maturation precedes the start of spermatogenesis and is coordinated, so efficient spermatogenesis will occur in the adults. The present study was conducted to evaluate endocrine regulation of germ and somatic cell homeostasis in the neonatal boar testis associated with the establishment of spermatogenesis. Testis tissue obtained from 3-, 5-, 7- and 14-day-old piglets were ectopically xenografted onto castrated, immunodeficient nude mice. Grafts were removed 22 weeks later and evaluated for growth and the establishment of spermatogenesis. Recipient mouse testosterone biosynthesis and follicle-stimulating hormone (FSH) concentrations were also assayed. Testis tissue graft growth was significantly greater in testis grafts from 3-day donor tissue when compared to all other ages; 5-, 7- and 14-day-old donor tissue weights were not significantly different at removal. Follicle-stimulating hormone concentrations in recipient mice supporting testis grafts from 5-, 7- and 14-day-old donor tissues did not differ and were similar to normal physiological levels in age-matched, intact nude mice. Serum FSH levels were significantly lower in recipient mice supporting testis grafts from 3-day-old donor tissue. Radioimmunoassay and biological assay indicated no differences in testosterone production by testis tissue grafts of varying donor age. Porcine testis tissue obtained from 3-, 5-, 7- and 14-day-old neonatal boars were all capable of producing round and elongate spermatids after 22 weeks of grafting, but testis grafts from 14-day-old donors had a significantly greater (eightfold) percentage of seminiferous tubules with spermatids compared to all other donor ages (p < 0.05). Cryopreservation did not affect the ability of testis tissue grafts to grow, produce testosterone or establish spermatogenesis when compared to controls (p < 0.05). Collectively, these data demonstrate intrinsic differences in the biological activity of germ and somatic cell populations during neonatal boar testis development associated with the establishment of spermatogenesis.

Free-Ranging Farm Cats: Home Range Size and Predation on a Livestock Unit In Northwest Georgia

This study’s objective was to determine seasonal and diurnal vs. nocturnal home range size, as well as predation for free-ranging farm cats at a livestock unit in Northwest Georgia. Seven adult cats were tracked with attached GPS units for up to two weeks for one spring and two summer seasons from May 2010 through August 2011. Three and five cats were tracked for up to two weeks during the fall and winter seasons, respectively. Feline scat was collected during this entire period. Cats were fed a commercial cat food daily. There was no seasonal effect (P > 0.05) on overall (95% KDE and 90% KDE) or core home range size (50% KDE). Male cats tended (P = 0.08) to have larger diurnal and nocturnal core home ranges (1.09 ha) compared to female cats (0.64 ha). Reproductively intact cats (n = 2) had larger (P < 0.0001) diurnal and nocturnal home ranges as compared to altered cats. Feline scat processing separated scat into prey parts, and of the 210 feline scats collected during the study, 75.24% contained hair. Of these 158 scat samples, 86 contained non-cat hair and 72 contained only cat hair. Other prey components included fragments of bone in 21.43% of scat and teeth in 12.86% of scat. Teeth were used to identify mammalian prey hunted by these cats, of which the Hispid cotton rat (Sigmodon hispidus) was the primary rodent. Other targeted mammals were Peromyscus sp., Sylvilagus sp. and Microtus sp. Invertebrates and birds were less important as prey, but all mammalian prey identified in this study consisted of native animals. While the free-ranging farm cats in this study did not adjust their home range seasonally, sex and reproductive status did increase diurnal and nocturnal home range size. Ultimately, larger home ranges of free-ranging cats could negatively impact native wildlife.

Acute ethanol exposure affects spermatogonial stem cell homeostasis in pre-pubertal mice.

Ethanol is a known modulator of neural stem cell development, but the consequences of ethanol toxicity on the cell fate decisions of spermatogonial stem cells (SSCs) is poorly understood. Using an in vivo treatment and stem cell transplantation approach, we investigated the effects of acute ethanol exposure on formation of the growing adult SSC population in neonatal and pre-pubertal mice. Treatment with a single dose of ethanol disrupted SSC homeostasis in vivo evidenced by a significant reduction (7-fold) of stem cell colonization efficiency in the testes of recipient mice following transplantation. Ethanol treatment also increased the rate of apoptosis in adult differentiating germ cells in situ. Gene expression analysis indicates that ethanol exposure has transient and long-term effects on the expression of GDNF and VEGF family molecules and supports the hypothesis that the niche microenvironment for SSCs is sensitive to ethanol toxicity during pre-pubertaland adult life.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Reproductive success in ruminants: A complex interaction among endocrine, metabolic, and environmental factors

The Physiology and Endocrinology Symposium titled “Reproductive Success in Ruminants: A Complex Interaction Between Endocrine, Metabolic and Environmental Factors” was held at the Joint Annual Meeting of the American Dairy Science Association and American Society of Animal Science (ASAS) from July 20 to 24, 2014, in Kansas City, MO. The symposium was organized by the Physiology and Endocrinology Committee, which included Gregoy Bedecarrats (University of Guelph, Ontario, Canada), Lance H. Baumgard (Iowa State University, Ames), Russell Hovey (University of California, Davis), G. Cliff Lamb (University of Florida, Marianna), José E. P. Santos (University of Florida, Gainesville), Brian K. Whitlock (University of Tennessee, Knoxville), and Kyle C. Caires (Berry College, Mount Berry, GA). The purpose of the symposium was to discuss new findings regarding mechanisms by which extrinsic and intrinsic factors can alter various aspects of endocrinology, metabolism, immunology, and physiology, each with important ramifications for reproductive success in a variety of ruminant models. Five outstanding scientists were selected by the symposium committee because they were each recognized as being exceptional speakers and leading experts in their respective fields. The symposium’s presentations included the ASAS–European Federation of Animal Science (EAAP) exchange presentation. In addition, 1 presentation was selected from among the standard submission of abstracts submitted by speakers who received an American Dairy Science Association–EAAP Travel Award. The first presentation of the symposium, titled “Recent advances in the hypothalamic control of reproduction,” was presented by Iain Clarke (Monash University, Clayton, Victoria, Australia). Reproduction cyclicity is driven by GnRH, and recent evidence indicates that kisspeptin neurons are most likely orchestrating the pulsatile secretion of GnRH in response to a variety of intrinsic and extrinsic factors (Clarke, 2014a). Whole-animal and cellular experiments conducted by Clarke’s laboratory significantly improved our understanding of the mechanisms by which leptin and other reproductive neuropeptides, such as neurokinin B and dynorphin, regulate reproductive cyclicity in the ewe in response to changes in metabolic status, lactation, and seasonality (Jacobi et al., 2013; Clarke, 2014b). Special emphasis was placed on understanding the dual role gonadotropin inhibitory hormone plays as a suppressor of reproductive function and as an appetite stimulant in neuropeptide Y neurons. Extensive in vivo and in vitro experimentation helped define novel relationships between metabolic circuits and reproductive circuits in the hypothalamus and, in doing so, highlight new signal transduction pathways important for the control of gonadotropin secretion in ruminants. The second speaker of the symposium was T. Welsh Jr. (Texas A&M University, College Station), who discussed the “Influence of stress on male reproductive physiology” (Welsh et al., 2014). It is known that environmental, psychological, and physiological stressors are associated with decreased fertility in domestic livestock species and humans. Recent work presented by the speaker demonstrated how stress horPHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Reproductive success in ruminants: A complex interaction among endocrine, metabolic, and environmental factors1