Ningxia Lu

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in the Testes of Male Mice Causes Subfertility, Reduces Sperm Numbers, and Alters Expression of Genes That Regulate Undifferentiated Spermatogonia

Vascular endothelial growth factor A (VEGFA) isoform treatment has been demonstrated to alter spermatogonial stem cell homeostasis. Therefore, we generated pDmrt1-Cre;Vegfa(-/-) (knockout, KO) mice by crossing pDmrt1-Cre mice to floxed Vegfa mice to test whether loss of all VEGFA isoforms in Sertoli and germ cells would impair spermatogenesis. When first mated, KO males took 14 days longer to get control females pregnant (P < .02) and tended to take longer for all subsequent parturition intervals (9 days; P < .07). Heterozygous males sired fewer pups per litter (P < .03) and after the first litter took 10 days longer (P < .05) to impregnate females, suggesting a more progressive loss of fertility. Reproductive organs were collected from 6-month-old male mice. There were fewer sperm per tubule in the corpus epididymides (P < .001) and fewer ZBTB16-stained undifferentiated spermatogonia (P < .003) in the testes of KO males. Testicular mRNA abundance for Bcl2 (P < .02), Bcl2:Bax (P < .02), Neurog3 (P < .007), and Ret was greater (P = .0005), tended to be greater for Sin3a and tended to be reduced for total Foxo1 (P < .07) in KO males. Immunofluorescence for CD31 and VE-Cadherin showed no differences in testis vasculature; however, CD31-positive staining was evident in undifferentiated spermatogonia only in KO testes. Therefore, loss of VEGFA isoforms in Sertoli and germ cells alters genes necessary for long-term maintenance of undifferentiated spermatogonia, ultimately reducing sperm numbers and resulting in subfertility.

VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance.

Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo.

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in Granulosa Cells Using pDmrt-1-Cre or Amhr2-Cre Reduces Fertility by Arresting Follicular Development and by Reducing Litter Size in Female Mice

Because VEGFA has been implicated in follicle development, the objective of this study was to determine the effects of granulosa- and germ cell-specific VEGFA loss on ovarian morphogenesis, function, and female fertility. pDmrt1-Cre mice were mated to floxed VEGFA mice to develop granulosa-/germ cell-specific knockouts (pDmrt1-Cre;Vegfa -/-). The time from mating to first parturition was increased when pDmrt1-Cre;Vegfa -/- females were mated to control males (P = 0.0008) and tended to be longer for heterozygous females (P < 0.07). Litter size was reduced for pDmrt1-Cre;Vegfa -/- females (P < 0.007). The time between the first and second parturitions was also increased for heterozygous females (P < 0.04) and tended to be increased for pDmrt1-Cre;Vegfa -/- females (P < 0.07). pDmrt1-Cre;Vegfa -/- females had smaller ovaries (P < 0.04), reduced plasma estradiol (P < 0.007), fewer developing follicles (P < 0.008) and tended to have fewer corpora lutea (P < 0.08). Expression of Igf1r was reduced (P < 0.05); expression of Foxo3a tended to be increased (P < 0.06); and both Fshr (P < 0.1) and Sirt6 tended to be reduced (P < 0.06) in pDmrt1-Cre;Vegfa -/- ovaries. To compare VEGFA knockouts, we generated Amhr2-Cre;Vegfa -/- mice that required more time from mating to first parturition (P < 0.003) with variable ovarian size. Both lines had more apoptotic granulosa cells, and vascular staining did not appear different. Taken together these data indicate that the loss of all VEGFA isoforms in granulosa/germ cells (proangiogenic and antiangiogenic) causes subfertility by arresting follicular development, resulting in reduced ovulation rate and fewer pups per litter.

Progressive obesity alters the steroidogenic response to ovulatory stimulation and increases the abundance of mRNAs stored in the ovulated oocyte

Obese women who are able to attain pregnancy are at increased risk for early‐pregnancy loss due, in part, to reduced oocyte quality. We and others have demonstrated that female Lethal Yellow (LY) mice and female C57BL/6 mice fed a high fat diet (B6‐HFD) exhibit phenotypes consistent with human obesity. These studies also showed that zygotes collected from LY and B6‐HFD females have reduced developmental competence. The current hypothesis is that LY and B6‐HFD females exhibit an abnormal response to gonadotropin stimulation compared to C57BL/6 controls fed normal rodent chow (B6‐ND), resulting in the ovulation of oocytes with an altered molecular phenotype which may contribute to its reduced developmental competence. To test this hypothesis, age‐matched B6‐ND, B6‐HFD, and LY females were stimulated with exogenous gonadotropins, then circulating hormone levels and the phenotypes of ovulated oocytes were analyzed. There was no difference in ovulation rate or in the percentage of morphologically abnormal oocytes collected from the oviduct of any females. Progesterone and progesterone/estradiol ratios, however, were increased in B6‐HFD and LY compared to B6‐ND females 16 hr post‐human chorionic gonadotropin treatment. The transcript abundance of several candidate oocyte genes was also increased in B6‐HFD‐ and LY‐derived oocytes compared to B6‐ND‐derived oocytes. These data suggest that increased insulin and leptin levels of obese females elevated circulating progesterone concentrations, altered transcriptional activity during oocyte growth, and/or impaired mechanisms of RNA translation and degradation during oocyte maturation. These changes in mRNA abundance likely contribute to reduced oocyte quality and the subsequent poor embryogenesis associated with obesity. Mol. Reprod. Dev. 81: 735–747, 2014.