Gloria I. Perez

A sperm ion channel required for sperm motility and male fertility

Calcium and cyclic nucleotides have crucial roles in mammalian fertilization, but the molecules comprising the Ca2+-permeation pathway in sperm motility are poorly understood. Here we describe a putative sperm cation channel, CatSper, whose amino-acid sequence most closely resembles a single, six-transmembrane-spanning repeat of the voltage-dependent Ca2+-channel four-repeat structure. CatSper is located specifically in the principal piece of the sperm tail. Targeted disruption of the gene results in male sterility in otherwise normal mice. Sperm motility is decreased markedly in CatSper-/- mice, and CatSper-/- sperm are unable to fertilize intact eggs. In addition, the cyclic-AMP-induced Ca2+ influx is abolished in the sperm of mutant mice. CatSper is thusxa0vital to cAMP-mediated Ca2+ influx in sperm, sperm motility and fertilization. CatSper represents an excellent target for non-hormonal contraceptives for both men and women.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals

Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter–reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.

Prolongation of ovarian lifespan into advanced chronological age by Bax -deficiency

Female mammals are endowed with a finite number of oocytes at birth, each enclosed by a single layer of somatic (granulosa) cells in a primordial follicle. The fate of most follicles is atretic degeneration, a process that culminates in near exhaustion of the oocyte reserve at approximately the fifth decade of life in women, leading to menopause. Apoptosis has a fundamental role in follicular atresia, and recent studies have shown that Bax, which is expressed in both granulosa cells and oocytes, may be central to ovarian cell death. Here we show that young adult female Bax–/– mice possess threefold more primordial follicles in their ovarian reserve than their wild–type sisters, and this surfeit of follicles is maintained in advanced chronological age, such that 20–22–month–old female Bax–/– mice possess hundreds of follicles at all developmental stages and exhibit ovarian steroid–driven uterine hypertrophy. These observations contrast with the ovarian and uterine atrophy seen in aged wild–type female mice. Aged female Bax–/– mice fail to become pregnant when housed with young adult males; however, metaphase II oocytes can be retrieved from, and corpora lutea form in, ovaries of aged Bax–/– females following superovulation with exogenous gonadotropins, and some oocytes are competent for in vitro fertilization and early embryogenesis. Therefore, ovarian lifespan can be extended by selectively disrupting Bax function, but other aspects of normal reproductive performance remain defective in aged Bax–/– female mice.

Antral Follicle Count Reliably Predicts Number of Morphologically Healthy Oocytes and Follicles in Ovaries of Young Adult Cattle

Abstract Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12–18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (≥25 follicles that were ≥3 mm in diameter) or low (≤15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.

Sphingosine 1-phosphate preserves fertility in irradiated female mice without propagating genomic damage in offspring

Sphingosine 1-phosphate preserves fertility in irradiated female mice without propagating genomic damage in offspring

Mitochondrial Rejuvenation After Induced Pluripotency

Background As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion “resets” some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells. Methodology/Principal Findings We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal. Conclusions/Significance These results — coupled with earlier data from our laboratory — suggest that IPSC conversion not only resets the “biological clock”, but can also rejuvenate the energetic capacity of derived cells.

Mitochondria and the death of oocytes.

In females of many species, over half of the germ-cell (oocyte) population dies by apoptosis before birth. For example, germ-cell numbers peak at 5–7×106 at week 20 of gestation in humans, but drop to less than 1×106 in the early neonatal period. Apparent germ-cell wastage occurs on a similar scale in female rodents, falling from 6.4×104 at day 17.5 of pregnancy to 1.9×104 shortly after birth. Krakauer and Mira have interpreted this death of germ cells as a developmental solution to the accumulation of mutations in mitochondria, proposing that prenatal oocyte apoptosis effectively removes oocytes carrying mutant mitochondria. Here we test whether mitochondria can actually influence oocyte fate by microinjecting small numbers of mitochondria into mouse oocytes and find that this prevents these cells from undergoing apoptosis. We also show that a common mitochondrial DNA deletion occurs more frequently in unfertilized, as compared with fertilized, human oocytes.

Reproductive biology: Mitochondria and the death of oocytes

In females of many species, over half of the germ-cell (oocyte) population dies by apoptosis before birth. For example, germ-cell numbers peak at 5–7×106 at week 20 of gestation in humans, but drop to less than 1×106 in the early neonatal period. Apparent germ-cell wastage occurs on a similar scale in female rodents, falling from 6.4×104 at day 17.5 of pregnancy to 1.9×104 shortly after birth. Krakauer and Mira have interpreted this death of germ cells as a developmental solution to the accumulation of mutations in mitochondria, proposing that prenatal oocyte apoptosis effectively removes oocytes carrying mutant mitochondria. Here we test whether mitochondria can actually influence oocyte fate by microinjecting small numbers of mitochondria into mouse oocytes and find that this prevents these cells from undergoing apoptosis. We also show that a common mitochondrial DNA deletion occurs more frequently in unfertilized, as compared with fertilized, human oocytes.

A central role for ceramide in the age-related acceleration of apoptosis in the female germline

An age‐dependent acceleration of apoptosis occurs in female germ cells (oocytes), and this requires communication between the oocyte and its surrounding somatic (cumulus) cells. Here we show in aged mice that ceramide is translocated from cumulus cells into the adjacent oocyte and induces germ cell apoptosis that can be prevented by sphingosine‐1‐phosphate. Trafficking of ceramide requires gap junction‐dependent communication between the cumulus cells and the oocyte as well as intact lipid rafts. Further, the occurrence of the elevated incidence of apoptosis in oocytes of aged females is concomitant with an enhanced sensitivity of the oocyte to a spike in cytosolic ceramide levels, as well as increased bax mRNA and Bax protein levels. Thus, the force driving the age‐related increase in female germ cell death is multifactorial, but changes in the intercellular trafficking of ceramide, along with hypersensitivity of oocytes to ceramide, are key factors in this process.